Glutamate Synthase: Structural, Mechanistic and Regulatory Properties, and Role in the Amino Acid Metabolism

Home , Amino acid synthesis, Glutamate synthase

Photosynthesis Research (2005) 83: 191–217 Springer 2005

Review Glutamate synthase: structural, mechanistic and regulatory properties, and role in the amino acid metabolism

Akira Suzuki1,* & David B. Knaﬀ2 1Unite´ de Nutrition Azote´e des Plantes, Institut National de la Recherche Agronomique, Route de Saint-Cyr, 78026 Versailles cedex, France; 2Department of Chemistry and Biochemistry, Texas Tech University, P.O. Box 41061, Lubbock, TX 79409-1061, USA; *Author for correspondence (e-mail: [email protected]; fax: +33-1-30833096)

Received 28 June 2004; accepted in revised form 20 September 2004

Key words: ammonium assimilation, glutamate synthase, glutamine synthetase, higher plants, nitrogen metabolism

Abstract Ammonium ion assimilation constitutes a central metabolic pathway in many organisms, and glutamate synthase, in concert with glutamine synthetase (GS, EC 6.3.1.2), plays the primary role of ammonium ion incorporation into glutamine and glutamate. Glutamate synthase occurs in three forms that can be distinguished based on whether they use NADPH (NADPH-GOGAT, EC 1.4.1.13), NADH (NADH-GO- GAT, EC 1.4.1.14) or reduced ferredoxin (Fd-GOGAT, EC 1.4.7.1) as the electron donor for the (two- electron) conversion of L-glutamine plus 2-oxoglutarate to L-glutamate. The distribution of these three forms of glutamate synthase in different tissues is quite specific to the organism in question. Gene structures have been determined for Fd-, NADH- and NADPH-dependent glutamate synthases from different organisms, as shown by searches in nucleic acid sequence data banks. Fd-glutamate synthase contains two electron-carrying prosthetic groups, the redox properties of which are discussed. A description of the ferredoxin binding by Fd-glutamate synthase is also presented. In plants, including nitrogen-fixing legumes, Fd-glutamate synthase and NADH-glutamate synthase supply glutamate during the nitrogen assimilation and translocation. The biological functions of Fd-glutamate synthase and NADH-glutamate synthase, which show a highly tissue-specific distribution pattern, are tightly related to the regulation by the light and metabolite sensing systems. Analysis of mutants and transgenic studies have provided insights into the primary individual functions of Fd-glutamate synthase and NADH-glutamate synthase. These studies also provided evidence that glutamate dehydrogenase (NADH-GDH, EC 1.4.1.2) does not represent a significant alternate route for glutamate formation in plants. Taken together, biochemical analysis and genetic and molecular data imply that Fd-glutamate synthase incorporates photorespiratory and non-photorespiratory ammonium and provides nitrogen for transport to maintain nitrogen status in plants. Fd-glutamate synthase also plays a role that is redundant, in several important aspects, to that played by NADH- glutamate synthase in ammonium assimilation and nitrogen transport.

Abbreviations: ASN – asparagine synthetase gene; CD – circular dichroism; EPR – electron paramagnetic resonance; DG – free energy change; DS – entropy change; FAD – ﬂavin adenine dinucleotide; Fd – ferredoxin; FMN – ﬂavin mononucleodide; Fe/S cluster – iron-sulfur cluster; FNR – ferredoxin: NADP+ oxidoreductase; GAT – glutamine amidotransferase; GDH – glutamate dehydrogenase; GOGAT – glutamate synthase; GLN1(2) – cytosolic (chloroplastic) glutamine synthetase gene; glsF – ferredoxin-glutamate synthase gene; GLT – NADH-glutamate synthase gene; gltB – NADPH-glutamate synthase a subunit 192 gene; gltD – NADPH-glutamate synthase b subunit gene; GltS – glutamate synthase; gltS – Fd-glutamate synthase gene; GLUI (2) – ferredoxin-glutamate synthase 1(2) gene; GS1(2) – cytosolic (chloroplastic/ plastidial) glutamine synthetase; NAR – nitrate reductase gene; NIR – nitrite reductase gene; Rubisco – ribulose-l,5-bisphosphate carboxylase/oxygenase

Introduction bacteria (Reitzer 1996). Both the Fd-glutamate synthase and NADH-glutamate synthase are Plants utilize inorganic nitrogen in the form of located in the chloroplast or plastid (Oliveira et al. ) + nitrate (NO3 ) and ammonium ion (NH4 ), when 1997). GS and glutamate synthase occur in mul- the latter is available in the soil or from the sym- tiple forms encoded by distinct genes (Lam et al. biotic fixation of atmospheric dinitrogen (N2) into 1996). Although there is some redundancy of + NH4 in root nodules of leguminous species. function among the multiple enzyme forms, for the Nitrate is reduced to nitrite, in a NAD(P)H- most part each form of GS and glutamate synthase dependent reaction, catalyzed by nitrate reductase plays a distinct physiological role in vivo during (NADH-NAR, EC 1.6.6.1; NAD(P)H-NAR, EC nitrogen absorption in roots, N2-fixation in root ) 1.6.6.2; NADPH-NAR, EC 1.6.6.3) in the cytosol. nodules, primary NO3 reduction, photorespira- + Nitrite is subsequently reduced to NH4 in a fer- tory nitrogen cycling and nitrogen translocation redoxin (Fd)-dependent reaction catalyzed by (Vance et al. 1994; Lam et al. 1996). Fd-dependent nitrite reductase (NIR, EC 1.6.6.4) An alternative pathway for the formation of in the chloroplast or plastid. Ammonium ion is the glutamate involves the reductive amination of + final form of inorganic nitrogen and the nitrogen 2-oxoglutarate by NH4 , catalyzed by mitochon- present in all organic nitrogen compounds, such as drial glutamate dehydrogenase (NADH-GDH, EC amino acids and nucleic acids, is derived from 1.4.1.2). However, the role of GDH in plant cells + NH4 (Lea et al. 1990). Ammonium is released remains controversial (Fox et al. 1995; Melo- and then re-assimilated during nitrogen mobiliza- Olivera et al. 1996; Miflin and Habash 2002). tion in germinating seeds, during the photorespi- Molecular genetic and biochemical studies using ratory conversion of glycine to serine in the cells of 13N- or 15N-radiorabeled tracers, enzyme inhibi- growing leaf, and during nitrogen remobilization tors, and mutants, as well as studies using trans- from sources to sinks (Ireland and Lea 1999). The genic plants affected in GS, glutamate synthase or + assimilation of NH4 into glutamine and gluta- GDH all indicate that the GS/glutamate synthase + mate is the crucial step in amino acid synthesis and cycle is the primary pathway for NH4 assimila- nitrogen metabolism. Glutamine synthetase (GS, tion (Ratcliffe and Shachar-Hill 2001; Lea and + EC 6.3.1.2) catalyzes the first step of NH4 Miflin 2004). Also, expression analysis revealed incorporation into glutamate using ATP to yield that the plants display cell-specific and organ- glutamine in the cytosol (GS1), in chloroplasts and specific patterns for expression of GS and gluta- in plastids (GS2). mate synthase genes by sensing the light and Glutamate synthase (glutamine: 2-oxoglutarate metabolite signals in the regulation of in vivo amidotransferase, henceforth abbreviated as either function of GS and glutamate synthase isoforms GOGAT or GltS) transfers the amide-nitrogen of (Edwards et al. 1990; Thum et al. 2003). The L-glutamine to 2-oxoglutarate, providing two amino-nitrogen of glutamate, incorporated into molecules of L-glutamate. Glutamate synthase in the carbon skeleton by the sequential reaction of plants is present in two distinct forms, one that GS and glutamate synthase, then serves as the uses reduced ferredoxin as the electron donor (Fd- source of the amino groups of aspartate and ala- GOGAT/Fd-GltS, EC 1.4.7.1) and one that uses nine, formed by the transamination of oxaloace- NADH as the electron donor (NADH-GOGAT/ tate and pyruvate, respectively (Reitzer 1996). As NADH-GltS, EC 1.4.1.14). A third form, which amino acid synthesis is controlled by availability uses NADPH as the electron donor (NADPH- of carbon skeletons, nitrogen assimilation is tightly GOGAT/NADPH-GltS, EC 1.4.1.13) is found in coupled to carbon metabolism. Glutamate, 193 aspartate and alanine then provide the nitrogen endosperms (Oaks et al. 1979), roots (Oaks et al. required for the formation of other amino acids. 1979), root nodules (Chen and Cullimore 1988) The amide-nitrogen of glutamine is used for the and cultured soybean cells (Chiu and Shargool biosynthesis of amino acids, including the forma- 1979). Although the results of these biochemical tion of asparagine from aspartate (Ireland and Lea studies suggest the presence of NADPH-glutamate 1999). Glutamine, asparagine, glutamate and synthase in these tissues, NADPH-glutamate syn- aspartate are the major amino acids in leaves and thase protein has not yet been unambiguously roots and are transported in the vascular tissues to identified in either photosynthetic or non-photo- control the nitrogen status during growth and synthetic tissues of any higher plant. It should also development of plants (Pate and Layzell 1990). be pointed out that no open reading frame coding In this review, we will analyze the current for NADPH-glutamate synthase has been detected information on the distribution of different types in the Arabidopsis genome database. of glutamate synthase in prokaryotes and Fd-glutamate synthase and NADH-glutamate eukaryotes. As reaction mechanisms and struc- synthase from the green alga Chlamydomonas tural aspects of glutamate synthase will be dis- reinhardtii have both been characterized (Galvań cussed in the accompanying article by Vanoni et al. 1984; Ma´rquez et al. 1984). Fd-glutamate et al., we will present instead a summary of the synthase has been detected in the chloroplast of current state of knowledge of the oxidation- green alga Caulerpa simpliciuscula (McKenzie reduction properties of Fd-glutamate synthase and et al. 1979). The plastid genome of red algae of its mode of interaction with ferredoxin. We will contains a structural gene of glsF (or gltB also describe the regulatory properties of gluta- according to the authors) for Fd-glutamate syn- mate synthase in higher plants with regard to thase in Antithamnion sp. (Valentin et al. 1993) defining the biological role of the enzyme in the and gltB in Porphyra purpurea (Reith and nitrogen assimilation and nitrogen translocation in Munholland 1993). Fd-glutamate synthase activity higher plants. has been detected in the cyanobacterium Syn- echococcus sp. PCC 6301 (Marque´s et al. 1992). It was reported that unicellular cyanobacterium Occurrence of glutamate synthases Synechocystis sp. PCC 6803 contains two putative Fd-glutamate synthase genes, gltB and glsF (later Two distinct classes of glutamate synthase are renamed gltS after it was confirmed that it encodes distinguished in higher plants: Fd-glutamate syn- a Fd-glutamate synthase) (Navarro et al. 1995). thase and NADH-glutamate synthase, both of However, the genome of Synechocystis sp. PCC which are located in the chloroplast or plastid. 6803 contains a sequence similar to the gltD Biochemical and molecular analyses detected both encoding the b-like subunit of bacterial NADPH- Fd-glutamate synthase and NADH-glutamate glutamate synthase (Kaneko et al. 1996). There- synthase in germinating seeds (Grevarec et al. fore, it may be that the gene originally thought to 2004), roots (Redinbaugh and Campbell 1993), encode a second Fd-glutamate synthase in Syn- root nodules (Chen and Cullimore 1988; Anderson echocysitis sp. PCC 6803 actually codes for the a et al. 1989; Vance et al. 1995; Cordovilla et al. subunit of ab heterodimeric glutamate synthase, 2000), cotyledons (Turano and Muhitch 1999), which is active with NADH as the electron donor etiolated shoots and leaves (Sakakibara et al. rather than with NADPH (Navarro et al. 2000). 1992a; Yamaya et al. 1992), green leaves (Sakaki- Both the glsF for Fd-glutamate synthase, and the bara et al. 1992b), cultured tobacco cells (Suzuki gltB and gltD have been cloned from the cyano- et al. 1982; Hayakawa et al. 1992). It has been bacterium Plectonema boryanum (Okuhara et al. reported that purified NADH-glutamate synthase 1999). The gltB and gltD encode the dissimilar a is either inactive with NADPH as an electron and b subunits, respectively, of NADH-glutamate donor (Hayakawa et al. 1992) or displays very low synthase (Okuhara et al. 1999). The complete activity (0.8–2.6% of the NADH-dependent genome of cyanobacterium Anabaena sp. PCC activity) (Chen and Cullimore 1988). NADPH- 7120 has been sequenced, showing that glsF linked glutamate synthase activity has been encoding Fd-glutamate synthase is the unique detected in seeds (Murray and Kennedy 1980), glutamate synthase gene (Martin-Figueroa et al. 194

2000), and to date genes for NADH-glutamate KODI glutamate synthase and the b subunit of the synthase have not been found in this cyanobacte- bacterial NADPH-glutamate synthase have been rium. In contrast, only NADH-glutamate synthase reported, but no genes with any significant has been detected in fungi and yeast such as homology to gltB have been detected in Neurospora crassa (Hummelt and Mora 1980), Pyrococcus sp. KOD1 (Jongsareejit et al. 1997). Saccharomyces cerevisiae (Cogoni et al. 1995; The Pyroccocus horikoshii OT3 genome contains a Filetici et al. 1996) and Kluyveromyces lactis sequence homologous to gltD (Kawarabayashi (Romero et al. 2000), where it appears to be et al. 1998). The presence of one of the gltB and present as a monomeric protein of high molecular gltD genes and the corresponding subunits in dif- weight. ferent archaea suggests that NAD(P)H-glutamate Bacterial NADPH-glutamate synthases are synthase of eubacteria and eukaryotes could have heterodimeric proteins. The structural gltB and originated from genes of the two different species gltD genes, coding for the large a subunit and (Dincturk and Knaff 2000). NADH-glutamate small b subunit, respectively, have been cloned synthase activity is found in animal cells (Sesha- from Escherichia coli (Oliver et al. 1987; Castan˜ o chalam et al. 1992; Hirayama et al. 1998; Dover- et al. 1992), Azospirillum brasilense (Pelanda et al. skog et al. 2000). A monomeric NADH-glutamate 1993), Salmonella typhimurium (Madonna et al. synthase of high molecular mass of 190–195 kDa 1985), Thiobacillus ferrooxidans (Deane and has been shown to be present in the body fat of the Rawling 1996), Rhizobium etli (Castillo et al. silkworm Bombyx mori (Hirayama et al. 1998). 2000). A different nomenclature has been used for the genes of Bacillus subtilis, where the names gltA and gltB have been used to designate the genes Gene and primary protein structure of glutamate encoding the a subunit and b subunit, respectively synthases (Belitsky et al. 1995). NADPH-glutamate synthase is also a ab heterodimeric protein in Aerobacter DNA sequences for genes and for cDNA have been aerogenes (Geary and Meister 1977), Bacillus characterized for Fd-glutamate synthase and magaterium (Hemmila¨ and Ma¨ ntsa¨ la¨ 1978), Rho- NADH-glutamate synthase from several plant dospirillum rubrun (Carlberg and Nordlund 1991), species, and those of Arabidopsis glutamate syn- and other bacteria (Brenchley et al. 1975; Okon thase are available from the complete sequence of et al. 1976; Smith et al. 1977; Ely et al. 1978; Arabidopsis genome. Fd-glutamate synthase genes Vanoni et al. 1990). However, NADH-glutamate (GLU, Fd-gltS) are homologous to gltB, which synthase activity has been detected in some nitro- codes for the a subunit of bacterial glutamate syn- gen-fixing bacteria (Nagatani et al. 1971) and in thase. In contrast, no homologies exist between three species of Chromatiaceae (Bast 1977). The Fd-glutamate synthase genes and gltD, the gene genomes of the archaeal bacteria Methanococcus encoding the b subunit in the ab protomer of bac- jannaschii (Bult et al. 1996) and Archaeoglobus terial NADPH-glutamate synthase (Table 1). In fulgidus (Klenk et al. 1997) both contain a Arabidopsis, Fd-glutamate synthase is encoded by sequence similar to the gltB for a bacterial two genes; GLU1 and GLU2, which are located on NADPH-glutamate synthase a subunit. These chromosome 5 and 2, respectively. The GLU1 archaeal sequences of approximately 500 amino cDNA has an ORF encoding a 1648-amino acid acid residues resemble the FMN-binding region precursor protein (180.1 kDa). It consists of a 131 - and C-terminal cysteine-rich region of the gltB- amino acid transit peptide (14.6 kDa) and a 1517- encoded a subunit of NADPH-glutamate synthase amino acid mature peptide (165.5 kDa) (Suzuki and and gltS-encoded Fd-glutamate synthase (Vanoni Rothstein 1997). Another reported cloning of and Curti 1999). Genomic DNA sequences similar GLU1 cDNA revealed a nearly identical sequence to the gltB and gltS have been identified in Met- except for the presence of a 75 bp sequence hanococcus thermoautotrophicum (Smith et al. (nucleotides 277–351, located between amino acids 1997). Another archaeal glutamate synthase has 94 and 122) that is likely to be an intron (Coschigano been cloned from Pyrococcus sp. KOD1, and this et al. 1998). By alignment with the cDNA sequence glutamate synthase protein has been expressed in of the GLU1 isoform, it has been determined that E. coli. Homologies between the Pyrococcus sp. the Arabidopsis GLU1 gene is transcribed as a 8590- 195

Table 1. Genes encoding glutamate synthases. Denomination of the gene and the protein of gene product corresponds to the term employed in the literature and in the GenBank database. Sequence information was obtained from the GenBank database using the accession number or loci indicated

Gene (acronym) Protein (acronym)

Higher plants GLU/glu/gluS/gltS Fd-glutamate synthase/Fd-GOGAT

Green leaves, etiolated leaves/shoots, roots, N2-ﬁxing nodules (M59190, Y09667, U03006, U39287, AY189525, AF039851, U39288, At5g04140, At2g41220) GLT NADH-glutamate synthase/NADH-GOGAT

Green leaves, etiolated leaves/shoots, roots, N2-ﬁxing nodules (L01660, L37606, AB008845: AB001916, AK110476, At5g53460) Algae glsF or gltB Fd-glutamate synthase/Fd-GOGAT Chlamydomonas reinhardtii (AF135592), Antithamnion sp. (Z21705, Z75242), Caulerpa simpliciuscula gltB Porphyra purpurea (U38804)

Cyanobacteria glsF or gltS Fd-glutamate synthase/Fd-GOGAT Synechocystis sp. PCC 6803 (X92480), Plectonema boryanum (D85735), Anabaena sp. PCC 7120 (AJ249913) gltB and gltD NADH-glutamate synthase/NADH-GOGAT Plectonema boryanum (D85230), Synechocystis sp. PCC 6803 (X80485)

Fungi gltBD NADH-glutamate synthase/NADH-GOGAT Neurospora crassa (XM328182, AL356815)

Yeasts GltBD, GLT1 NADH-glutamate synthase/NADH-GOGAT Saccharomyces cerevisiae (X89221)

Bacteria gltB and gltD NADH-glutamate synthase/NADPH-GOGAT/NADPH-GltS Escherichia coli (M18747, L20253, M68876), Azospirillum brasilense (AF192408, X71090), Pseudomonas aeruginosa PA01 (AE004916), Preudomonas aeruginosa (U81261, AE004916), Salmonella typhimurium (AE008853), Thiobacillus ferrooxidans (U36427), Rhizobium etli (AF107264), Chromatiaceae gltA and gltB NADPH-glutamate synthase/NADPH-GOGAT/NADPH-GltS Bacillus subtilis (M28509)

Archaeal bacteria gltB NADPH-glutamate synthase/NADPH-GOGAT/NADPH-GltS Methanococcus jannaschii (U67575), Archaeoglobus fulgidus (AE001038), Methanococcus thermoautotrophicum (AE000800), gltD or gltA Pyrococcus sp. KOD1, Pyroccocus horikoshii OT3 (PH1873)

Insect gltBD NADH-glutamate synthase/NADH-GOGAT/NADH-GltS silkworm Bombyx mor Higher plants/M59190: Zea mays Fd-glutamate synthase mRNA; L01660: Medicago sativa NADH-glutamate synthase mRNA; L37606: Medicago sativa NADH-glutamate synthase gene; U03006: Spinacia aleracea Fd-glutamate synthase mRNA; Y09667: Arabidopsis Fd-glutamate synthase GLU1 mRNA; U39287: Arabidopsis Fd-glutamate synthase GLU1 mRNA; AY189525: Arabidopsis Fd-glutamate synthase GLU1 gene; At5g04140: Arabidopsis Fd-glutamate synthase GLU1 gene; AF039851: Glycine max: Fd-glutamate synthase glu mRNA; U39288: Arabidopsis Fd-glutamate synthase GLU2 mRNA; At2g41220: Arabidopsis Fd-glutamate synthase GLU2 gene; At5g53460: Arabidopsis NADH-glutamate synthase GLT1 gene; AB008845: Oryza sativa NADH-glutamate synthase mRNA; AB001916: Oryza sativa NADH-glutamatee synthase gene; AK110476: Oryza sativa cDNA clone (002-166-H10) – Algae/AF135592: Chlamydomonas reinhardtii putative Fd-glutamate synthase gene; Z75242: Antitamnion sp. glutamate synthase gltB gene; Z21705: Antitamnion sp. chloroplast Fd-glutamate synthase gltB gene – Cyanobacteria/X92480: Synechocystis sp. Fd-glutamate synthase gltS gene; X80485: Synechocystis sp. Fd-glutamate synthase gltB gene; D78371: Synechocystis sp. putative Fd-glutamate Continued overleaf 196 synthase gene; D85735: Plectonema boryanum Fd-glutamate synthase glsF gene; D85230: Plectonema boryanum URF141 NADH- glutamate synthase gltB and gltD genes; AJ249913: Anabaena sp. PCC 7120 Fd-glutamate synthase gltS gene – Fungi/XM328182: Neurospora crassa OR74A putative glutamate synthase mRNA; AL356815: Neurospora crassa putative NADPH-glutamate synthase glt1 gene (BAC clone B24H17) – Yeast/X89221: Saccharomyces cerevisiae glutamate synthase gene – Bacteria/M18747: Escherichia coli glutamate synthase gltB and gltD genes; L20253: Escherichia coli glutamate synthase gltB gene; M68876: Escherichia coli glutamate synthase gltB gene; AF192408: Azospirillum brasilense NADPH-glutamate synthase gltB and gltD genes; X71090: Azospirillum brasilense NADPH-glutamate synthase gltB gene; U81261: Preudomonas aeruginosa NADPH-glutamate synthase gltB and gltD genes; AE004916: Preudomonas aeruginosa PA01 NADPH-glutamate synthase gltD gene; AE008853: Saimonella typhimurium LT2 glutamate synthase gltD gene (complete genome, section 157 of 220); U36327: Thiobacillus ferrooxidants glutamate synthase gltB and. gltD genes; AF107264: Rhizobium etli glutamate synthase gltB and gltD genes; M28509: Bacillus subtilis glutamate synthase gltA gene – Archaeal bacteria/U67575: Methanococcus jannaschii NADPH-glutamate synthase gltB gene; AE001038: Archaeoglobus fulgidus DSM 4304 glutamate synthase gltB gene; AE000800: Methanobacterium thermoautotrophicum genome glutamate synthase gltB gene; PH 1873: Pyrococcus horikoshii glutamate synthase gltD gene. nucleotide mRNA, which consists of a 5¢-untrans- mate synthase has an ORF encoding a 1616-amino lated region (5¢-UTR) (179 nucleotides), a coding acid precursor peptide (174.7 kDa). The mature sequence (8154 nucleotides) and a 3¢-UTR (257 protein consists of 1519 amino acids with a molec- nucleotides) (unpublished data and At5g04140). ular mass of 165.3 kDa (Sakakibara et al. 1991). The transcribed region of GLU1 is composed of 33 The mature form of spinach Fd-glutamate synthase exons interrupted by 32 introns (Figure 1). The has been shown to contain 1504 amino acids and the cDNA of the GLU2 isoform has an ORF which mature forms of the maize and spinach Fd-gluta- encodes a 1629-amino acid precursor peptide mate synthases are 83% identical at the amino acid (177.8 kDa) (Coschigano et al. 1998). The GLU2 level (Nalbantoglu et al. 1994; Dincturk and Knaﬀ gene of 10728 nucleotides contains a 5¢-UTR (274 2000). These Fd-glutamate synthase peptides share nucleotides) and the coding sequence includes 33 a signiﬁcant similarity (40–42%) with the gltB-en- exons and 32 introns (Figure 1). The predicted coded a subunit (166 kDa), but contain no amino acid sequences of GLU1 and GLU2 isoforms regions similar to the gltD-encoded b subunit are 80% identical. The cDNA for maize Fd-gluta- (52 kDa) of the (ab)x heterodimeric form of bac-

GLU1 for Fd-GOGAT (At5g04140)

1 2000 4000 6000 8000

GLU2 for Fd-GOGAT (At2g41220)

1 2000 4000 6000 8000 10000 bp

GLT for NADH-GOGAT (At5g54360)

1 2000 4000 6000 8000 bp Figure 1. Diagrammatic structure of the genes for ferredoxin-glutamate synthase (GLU1 and GLU2) and NADH-glutamate synthase (GLT)inArabidopsis thaliana. Exons are represented by boxes and introns are indicated by lines. Numbers start from the first nucleotide of the coding sequence. Chromosomal map position of the glutamate synthase genes is denoted by line at right of the structural presentation. 197 terial NADPH-glutamate synthase (Table 1). In the (Avila et al. 1993) and grapevine (Loulakakis and red alga Antithamninon sp., glsF (gltF) encodes a Roubelakis-Angelakis 1997). On the other hand, Fd-glutamate synthase of 1537 amino acids, corre- a single Fd-glutamate synthase gene is identified sponding to the gltB-encoded a subunit of bacterial in maize (Sakakibara et al. 1991) and spinach enzymes (Valentin et al. 1993). Fd-glutamate syn- (Nalbantoglu et al. 1994). thase from cyanobacterium Synechococcus sp. PCC Plant NADH-glutamate synthase genes (GLT, 6301 has a molecular mass of 160 kDa (Marque´s NADH-gltS) are single open reading frames, with 5¢ et al. 1992). The gltB and gltS of the cyanobacte- ends that show homology to gltB and 3¢ ends that rium Synechocystis sp. PCC 6803 encode an ORF of are homologous to the gltD of bacterial NADPH- 1550 amino acids (169.0 kDa) and 1557 amino acids glutamate synthase. The GLT gene in alfalfa nod- (170 kDa), respectively. The amino acid sequence ules has a transcribed region (12214 nucleotides analyses showed that two glutamate synthase composed of 22 exons and 21 introns), which polypeptides contain a conserved peptide loop encodes 2194 amino acids (240.4 kDa) and includes insert unique to Fd-glutamate synthase (Vanoni a 291-nucleotide 5¢UTR and a 101-amino acid and Curti 1999). Both the glsF for Fd-glutamate presequence (Gregerson et al. 1993; Vance et al. synthase, and the gltB and gltD for NADH-gluta- 1995). The rice NADH-glutamate synthase GLT is mate synthase have been cloned from the cyano- composed of a 5¢UTR (261 nucleotides), a coding bacterium Plectonema boryanum (Okuhara et al. sequence (6498 nucleotides) and a 3¢UTR (285 1999). The glsF encodes an ORF of 1551 amino nucleotides) (Goto et al. 1998). It contains 23 exons acids (169 kDa). The complete nucleotide sequence and 22 introns, and an ORF encodes 2166 amino of 9038 bp of Plectonema boryanum genome was acids (236.7 kDa), preceded by a 99-amino acid characterized (Okuhara et al. 1999), and it contains presequence. The exon/intron organization of the two ORF encoding polypeptides of 1530 amino rice gene is similar to that found in the transcribed acids (168 kDa) (GltB) and 492 amino acids portion of the alfalfa NADH-glutamate synthase (54 kDa) (GltD) with 106 bp apart. Despite the lack gene, but the alfalfa gene appears to contain an of similarity of the NH2-terminal signal peptides of additional exon lacking in the rice gene. A partially different Fd-glutamate synthases (105 amino acids/ transcribed region of the Arabidopsis NADH-glu- Arabidopsis GLU1; 107 amino acids/Arabidopsis tamate synthase gene (9112 nucleotides) is com- GLU2; 97 amino acids/maize GLU), they have the posed of 20 exons and 19 introns (Figure 1). The common characteristics of chloroplast transit pep- exons encode a precursor peptide predicted to tides, showing high contents of basic amino acids, contain 2208 amino acids (241.9 kDa). The exon/ hydroxylated amino acids and small hydrophobic intron organization is similar to that of the GLT amino acids such as alanine and valine and a high genes of alfalfa and rice. Most of the corresponding serine/arginine ratio (Sakakibara et al. 1991; introns of rice and alfalfa NADH-glutamate syn- Suzuki and Rothstein 1997). The predicted molec- thase genes show 20–40% similarity (Goto et al. ular masses of mature proteins, which start with a 1998). NADH-glutamate synthase GLT contains cysteine residue, are similar to the values calculated the conserved sequences of gltB and gltD found in from the mobility of the polypeptide by SDS-PAGE prokaryotic NADPH-glutamate synthase genes, (Sakakibara et al. 1991; Suzuki and Rothstein 1997; and a b subunit-like polypeptide has been fused at Dincturk and Knaff 2000). Partial Fd-glutamate the C-terminus of the a subunit-like polypeptide. synthase cDNAs have also been determined from NADH-glutamate synthase has also been shown to other plant species including tobacco (Zehnacker be a monomeric polypeptide in fungi (Hummlet and et al. 1992), barley (Avila et al. 1993), spinach Mora 1980), yeasts (Cogoni et al. 1995; Filetici (Nalbantoglu et al. 1994), alfalfa nodules (Vance et al. 1996; Romero et al. 2000) and insects et al. 1995), pine (Garcıá-Gutie´rrez et al. 1995), (Hirayama et al. 1998). A region linking heterodi- grapevine (Loulakakis and Roubelakis-Angelakis meric ab subunit-like polypeptides of NADH-glu- 1997) and soybean (Turano and Muhitch 1999). tamate synthase contains hydrophilic and charged Although definite evidence is not yet available, amino acids (histidine, lysine, arginine, glutamate) Fd-glutamate synthase may be encoded by two (Gregerson et al. 1993; Goto et al. 1998). Arabid- genes in tobacco (Nicotiana tabacum, amphidiploid) opsis NADH-glutamate synthase has characteris- (Zehnacker et al. 1992), barley (on chromosome 2) tics of a plastid-targeting 166 amino acid 198 presequence (a high serine/arginine ratio, and high et al. 1993). NADPH-glutamate synthase is cata- asparagine and glutamate contents) while the alfalfa lytically active as an ab protomer of approximately NADH-glutamate synthase leader sequence pre- 200 kDa, and the NADPH-glutamate synthase dicts a mitochondrial localization (Gregerson et al. holoenzyme appears to be an (ab)4 tetramer (Stabile 1993). It has been proposed that the pre-protein of et al. 2000; Petoukhov et al. 2003). Kinetic and unusual amino acid composition undergoes a mul- mechanistic properties were extensively studied for tiple processing prior to the import of the mature bacterial NADPH-glutamate synthase and cyano- protein into the plastid: a first cleavage upon bacterial and plant Fd-glutamate synthases translocation across plastid membrane and a sec- (Vanoni et al. 2005). ond cleavage to produce the mature protein starting with the cysteine residue, which is also found as the Amidotransferase and synthase reactions of first amino acid of bacterial glutamate synthases glutamate synthases (Gregerson et al. 1993). Several lines of evidence indicate that dual targeting of nuclear gene-encoded Fd-glutamate synthase is active as a bacterial pre-proteins, including the GS2 precursor to the a subunit-like single polypeptide in which non- chloroplasts and to the mitochondria, can occur covalently bound reduced Fd provides the elec- (Taira et al. 2004). All the available evidence indi- trons for the formation of L-glutamate from cates that the alfalfa NADH-glutamate synthase is L-glutamine and 2-oxoglutarate. NADPH-gluta- located in the amyloplast (Trepp et al. 1999b). mate synthase is active as an (ab)x heterodimer in Yeast NADH-glutamate synthase is preceded by a which NADPH binds to the b subunit and delivers transit peptide of 53 amino acids. This presequence the electrons for the reductive formation of l-glu- is supposed to serve as a reserve of inactive gluta- tamate in the a subunit. Structural studies, largely mate synthase that could be activated by the cleav- by X-ray crystallography, have provided the three- age and the consequent exposure of N-terminal dimensional structures of the NADPH-glutamate cysteine of the mature protein in the cell (Filetici synthase a subunit from Azospirillum brasilense et al. 1996). NADH-glutamate synthase may be (Binda et al. 2000) and Fd-glutamate synthase encoded by a small gene family in Alfalfa nodules from Synechocystis sp. PCC 6803 (van den Heuvel (Gregerson et al. 1993), while it is encoded by a et al. 2002, 2003; Vanoni et al. 2005). Structure- single gene in Arabidopsis (Lam et al. 1996) and rice based biochemical analyses have characterized the (Goto et al. 1998). catalytic mechanisms of the complex iron-sulfur Prokayotic NADPH-glutamate synthase is flavoproteins of Fd-glutamate synthase and encoded by the gltB and gltD genes for the large a NADPH-glutamate synthase. L-Glutamine- subunit (»150 kDa) and small b subunit (»50 kDa), dependent amidotransferase activity takes place respectively (Table 1). The structural genes of gltB on the N-terminal glutamine amidotransferase and gltD were initially cloned from the genomic (GAT) domain. This amidotransferase domain DNA of E. coli (Oliver et al. 1987), Azospirillum belongs to the Pur-F-type amidotransferases class, brasilense (Pelanda et al. 1993) and other bacteria now defined as NH2-terminal nucleophile (Ntn)- (as noted above, gltA has been used to denote the type class, and corresponds to the N-terminal 450 gene coding for the a subunit and gltB to denote the residues of Fd-glutamate synthase, NADH-gluta- gene coding for the b subunit in the Bacillus subtilis mate synthase and NADPH-glutamate synthase protein (Belitsky et al. 1995). In E. coli, the terminal (Oliver et al. 1987;Gregerson et al. 1993; Pelanda codon TAA of the gltB for the a subunit and the et al. 1993; Navarro et al. 1995; Suzuki and translation initiation site of the gltD genes for the b Rothstein 1997; Coschigano et al. 1998; Vanoni subunit are separated by a 12 nucleotide-intercis- and Curti 1999). The cysteine 1 residue of the tronic DNA region and form an operon with gltF GAT domain is conserved as the first amino acid encoding a putative kinase (Oliver et al. 1987; of the mature form of Fd-glutamate synthase and Castan˜ o et al. 1992). The gltB gene is upstream of of NADH-glutamate synthase, and this domain is the gltD gene in the glt operon (gltBDF)inE. coli, involved in the release of glutamine-amide group but the organization of the glt locus in Azosprillum and formation of an enzyme-c-glutamyl thioester brasilense is the opposite (Madonna et al. 1985; intermediate prior to glutamate formation in the a Oliver et al. 1987; Castan˜ o et al. 1992; Pelanda subunit. On addition of ammonia from L-gluta- 199 mine amide group, 2-oxoglutarate is converted to 1995; Suzuki and Rothstein 1997). As FAD is not 2-iminog1utarate intermediate at the FMN/FeS present in the Fd-glutamate synthase (Hirasawa synthase site. In GLU1-encoded Fd-glutamate et al. 1996) and recombinant NADPH-glutamate synthase of Arabidopsis, a region of 52 amino synthase a subunit (Vanoni et al. 1998), it has acids (leucine 1079–threonine 1130 of the mature been suggested that this putative ADP-binding protein) belonging to the FMN binding domain is fold may serve for the binding of a regulatory located on the exons 20 and 21 (Suzuki and adenylate-containing nucleotide. Recently, three- Rothstein 1997 and unpublished data). Aspartate dimensional structural analysis of NADPH-glu- 1100 and lysine 1104, which bind to the ribityl side tamate synthase a subunit (Binda et al. 2000) and chain of FMN are conserved in many Fd-gluta- of Fd-glutamate synthase (van den Heuvel et al. mate synthases, NADH-glutamate synthases and 2002) clearly showed that this region is part of the NADPH-glutamate synthases (Oliver et al. 1987; C-terminal b-helical domain of glutamate synthase Sakakibara et al. 1991; Gregerson et al. 1993; which appears to serve a structural rather than a Pelanda et al. 1993; Navarro et al. 1995; Suzuki catalytic or ligand binding role. Two regions and Rothstein 1997). Arabidopsis Fd-glutamate matching the consensus sequence for the forma- synthase contains three cysteines (cysteine 1132, tion of adenylate-binding folds are found in the cysteine 1138 and cysteine 1143) (Suzuki and C-terminal part of NADH-glutamate synthase. Rothstein 1997), which are located on the exon 21. Five conserved glycine residues (glycine 1974, This cysteine cluster exhibits (CX5CX4C) spacing glycine 1976, glycine 1979, glycine 1990 and glu- and aligns with the similar cysteine residues of all tamate 1998) are on exon 20 of alfalfa NADH- Fd-glutamate synthases, NADPH-glutamate syn- glutamate synthase (Vance et al. 1995) or the thases and NADH-glutamate synthases, with the equivalent five residues on exon 21 of rice NADH- three cysteine residues serving as ligands to the glutamate synthase (Goto et al. 1998). This region single [3Fe-4S] 1+,0 center present in the enzyme is involved in NADH-binding and it finds no (Knaff et al. 1991; Binda et al. 2000; van den counterpart in the eukaryotic Fd-glutamate syn- Heuvel et al. 2002). According to the structure- thase. based studies, the FMN/FeS synthase site medi- ates the transfer of the reducing equivalents Redox properties through an intramolecular electron transfer chain connecting the initial electron donor and Oxidation-reduction titrations of spinach Fd-glu- synthase site, and leads to the 2-iminoglutarate tamate synthase, using absorbance changes in the reduction and L-glutamate formation (Hirasawa visible region to monitor the redox state of the et al. 1996; Vanoni and Curti 1999; van der FMN group and changes in electron paramagnetic Heuvel et al. 2004). Fd-glutamate synthases have resonance (EPR) spectra to monitor the redox a short polypeptide-insert conserved in the syn- state of the [3Fe-4S]0,+1 cluster allowed measure- thase domain (Vanoni and Curti 1999), and it has ment of the oxidation-reduction midpoint poten- been suggested that a single Fd binds to the tial (Em) of both prosthetic groups at pH 7.7, the enzyme surface in the vicinity of the [3Fe-4S] pH-optimum for the enzyme. Titrations of the cluster and the FMN cofactor (see below) and that [3Fe-4S] cluster gave an excellent fit to the Nernst sequential one-electron transfers from first one Equation for a one-electron redox couple with an reduced ferredoxin and then from a second Em value of )170 ± 10 mV and the FMN titra- reduced ferredoxin provide, via these prosthetic tion gave an excellent fit to the Nernst Equation groups, the two electrons needed to reduce 2-imi- for a two-electron redox couple with an Em value noglutarate (van den Heuvel et al. 2003). The of )180 ± 10 mV (Hirasawa et al. 1992). Thus, C-terminal region of Fd-glutamate synthase con- within the experimental uncertainties of the mea- tains one glycine-rich region (running from glycine surements, the two prosthetic groups of the 1389 through glycine 1434 of GLU1-encoded enzyme are isopotential. Absorbance spectra in the Fd-glutamate synthase from Arabidopsis), which visible region, taken over the course of the FMN showed a limited sequence similarity to the con- titration, showed no evidence for detectable sensus sequence for the formation of an adenylate amounts of the one-electron reduced FMN sem- binding site (Pelanda et al. 1993; Navarro et al. iquinone (Hirasawa et al. 1992). This observation, 200 and the fact that no free radical signals attribut- approximately isopotential (i.e., with Em values of able to a FMN semiquinone were detected in the )240 mV for the FMN and )260 mV for the [3Fe- course of the EPR measurements (Hirasawa et al. 4S] cluster) in the a-subunit of the NADPH- 1992), are both consistent with two-electron dependent glutamate synthase from A. brasilense character of the titration curve. A cyclic voltam- (Ravasio et al. 2001, 2002). However, given the metry investigation of the redox properties of experimental uncertainties in these measurements spinach glutamate synthase also demonstrated and the fact that Em values are quite sensitive to that the two prosthetic groups of the enzyme are experimental conditions (such as pH, temperature isopotential, although this technique gave a and ionic strength), it is perhaps more important to somewhat more negative Em value of )225 mV for focus on the similarities in these Em values, rather both groups (Hirasawa et al. 1996). Thus, it was than on relatively small differences. It should also be not possible to predict the likely sequence of mentioned that titration of Synechocystis sp. PCC electron transfer events between the two prosthetic 6803 Fd-glutamate synthase with L-glutamate un- groups of spinach Fd-glutamate synthase on der anaerobic conditions caused an initial reduction thermodynamic grounds alone. It is known that of the [3Fe-4S] cluster followed by a subsequent complex formed between spinach ferredoxin and reduction of FMN (Ravasio et al. 2002), consistent spinach FNR causes shifts in the Em values of both with a more positive Em value for the [3Fe-4S] the [2Fe-2S] cluster of ferredoxin and the FAD cluster. Similar results were found with A. brasilense group of FNR (Knaff 1996). Thus, although no NADPH-dependent glutamate synthase a subunit, evidence is currently available for any such shifts while only FMN was reduced during L-glutamate in redox potentials arising from the interaction equilibrium titrations of the NADPH-glutamate between ferredoxin and Fd-glutamate synthase, synthase (ab)x holoenzyme (Ravasio et al. 2001, the Em values measured for Fd-glutamate synthase 2002). More detailed descriptions of the redox alone may perhaps be different from the values properties of Synechocystis sp. PCC 6803 Fd-glu- operating within a Fd-glutamate synthase complex tamate synthase and of the A. brasilense NADPH- with ferredoxin. glutamate synthase are presented in the article by Oxidation-reduction titrations of the FMN in Vanoni et al. in this issue. Synechocystis sp. PCC 6803 Fd-glutamate synthase gave a good fit to the two-electron Nernst Equation Complex formation with ferredoxin with Em ¼ )200 ± 25 mV at pH 7.5 (Navarro et al. 2000). As was the case for the spinach enzyme, A large body of evidence supports the hypothesis no evidence for the presence of a flavin semiquinone that ferredoxin forms electrostatically-stabilized was observed in the course of titrations of the FMN complexes with enzymes that use ferredoxin as the group of Synechocystis sp. PCC 6803 Fd-glutamate electron donor, with ferredoxin supplying most of synthase, suggesting that the two one-electron Em the negative charges involved in complex forma- values for the oxidized/semiquinone and semiqui- tion and the target enzyme supplying most of the none/fully-reduced FMN couples differ by at least positive charges (Knaff 1996). Spectral perturba- 100 mV (Ravasio et al. 2002). An estimation of the tions, gel filtration chromatography, membrane 0,+1 Em value of the [3Fe-4S] cluster in Synechocystis ultrafiltration and chemical cross-linking experi- sp. PCC 6803 Fd-glutamate synthase from absor- ments are all consistent with the hypothesis that bance changes in the visible region during an ferredoxin and Fd-glutamate synthase form such a anaerobic titration of the enzyme with sodium complex (Knaff 1996). Changes in the UV/visible dithionite suggested that the Em value of the cluster spectra of spinach ferredoxin and/or spinach was 40–50 mV more positive than that of the Fd-glutamate synthase, that occur when the two enzyme’s FMN group, putting the Em of the [3Fe- proteins are mixed and which arise from complex 4S]0,+1 cluster in Synechocystis sp. PCC 6803 formation between the two proteins, only occur at Fd-glutamate synthase at the rather positive value low ionic strength, consistent with the idea that of )150 to )160 mV (Ravasio et al. 2002). This electrostatic forces play a significant role in stabi- result was somewhat surprising, given the fact that lizing the spinach chloroplast ferredoxin/gluta- the two prosthetic groups are not only isopotential mate synthase complex (Hirasawa et al. 1986). The in spinach Fd-glutamate synthase, but are also observation that the two spinach chloroplast pro- 201 teins co-migrate during gel filtration chromatog- cells of the cyanobacterium Anabaena sp. PCC raphy at low ionic strength but not at high ionic 7120 has kinetic and binding parameters for strength is also consistent with the formation of an spinach Fd-glutamate synthase very similar to electrostatically-stabilized complex between spin- those measured for spinach ferredoxin and the ach ferredoxin and spinach Fd-glutamate synthase availability of a number of site-specific variants of (Hirasawa et al. 1986). Analysis of the hyperbolic this very well-characterized cyanobacterial ferre- plots of the magnitude of the absorbance changes doxin, for which a high-resolution X-ray crystal arising from complex formation between spinach structure is available (Rypniewski et al. 1991; ferredoxin and spinach Fd-glutamate synthase Holden et al. 1994; Hurley et al. 1997), led to its versus ferredoxin concentration indicated a single use in such studies (Hirasawa et al. 1998). binding process with a Kd of 14.5 lM at low ionic Replacement of a highly-conserved ferredoxin strength (Hirasawa et al. 1986). It should also be glutamate residue near the C-terminus of the mentioned that the rates of the ferredoxin-depen- protein (Glu94 in Anabaena sp. PCC 7120 ferre- dent reactions catalyzed by both Synechocystis sp. doxin) by either lysine or glutamine produced a PCC 6803 Fd-glutamate synthase (Schmitz et al. very large decrease in the ability of the ferredoxin 1996) and spinach Fd-glutamate synthase to serve as an efficient electron donor to spinach (M. Hirasawa, unpublished observations) decline Fd-glutamate synthase (Hirasawa et al. 1998). In markedly with increasing ionic strength, consistent contrast, similar replacements at the adjacent with the idea that electrostatic interactions be- glutamate, Glu95, had essentially no effect on tween the proteins play an important role at some ferredoxin-dependent activity of the reaction cat- point in the reaction catalyzed by Fd-glutamate alyzed by spinach Fd-glutamate synthase synthase. (Hirasawa et al. 1998). This sort of high positional Chemical modification of spinach Fd-gluta- specificity, when comparing effects of modifying mate synthase with either the lysine-modifying these two adjacent glutamate residues, had been reagent, N-acetylsuccinimide, or the arginine- observed previously in several studies of the modifying reagent, phenylglyoxal, results in inhi- interaction between ferredoxin and FNR and bition of enzyme activity when reduced ferredoxin between ferredoxin and ferredoxin-dependent serves as the electron donor but had no inhibitory nitrite reductases (Knaff 1996). Similar site-direc- effect on enzyme activity with the non-physiolog- ted mutagenesis studies indicate that Glu94 in ical electron donor, reduced methyl viologen Anabaena sp. PCC 7120 ferredoxin plays an (Hirasawa and Knaff 1993). These observations, important role in the interaction between ferre- and the fact that formation of the ferredoxin/glu- doxin and the Synechocystis sp. PCC Fd-gluta- tamate synthase complex prior to addition of mate synthase but, as site-specific replacements of either chemical modifier completely protected the the adjacent Glu95 were not a part of this study, enzyme from inhibition are consistent with a role no conclusions could be drawn about any posi- for lysine and arginine residues in ferredoxin- tional specificity (Schmitz et al. 1996). In contrast binding by the enzyme (Hirasawa and Knaff 1993). to the differences observed in replacing of Glu94 If the ferredoxin-binding site of spinach versus replacing Glu95 in Anabaena sp. PCC 7120 Fd-glutamate synthase does indeed contain posi- ferredoxin on the ability of reduced ferredoxin to tively-charged arginine and lysine residues, one serve as an electron donor for spinach Fd-gluta- would expect that ferredoxin would contribute mate synthase, a study with C. reinhardtii negatively-charged residues to electrostatic inter- Fd-glutamate synthase showed that elimination of actions involved in complex formation. In fact, it the negative charge on Glu92 of C. reinhardtii has been shown that chemical modification of ferredoxin (the residue that corresponds to Glu95 carboxylic acid side chains of spinach ferredoxin in Anabaena sp. PCC 7120 ferredoxin) by site- with glycine ethyl ester and a water-soluble car- specific mutagenesis produces a significant bodiimide (a treatment that elements the negative decrease in the rate of the reaction catalyzed by charges on glutamate and aspartate residues) C. reinhardtii Fd-glutamate synthase, although the decreases the binding affinity of ferredoxin for extent of inhibition (compared to the rate obtained spinach Fd-glutamate synthase (Hirasawa et al. with wild-type C. reinhardtii ferredoxin) was 1986). The wild-type ferredoxin from vegetative somewhat less than that observed when the nega- 202 tive charge on Glu91 of C. reinhardtii ferredoxin oriented water molecules, bound to the protein (the residue that corresponds to Glu94 in Anaba- surfaces, from the protein/protein interface region ena sp. PCC 7120 ferredoxin) was eliminated by into the bulk solvent (Jelesarov and Bosshard site-specific mutagenesis (Garcıá-Sańchez et al. 1994). A similar situation appears to be the case 2000). These studies also indicated that a second for the complex between the two Anabaena sp. acidic region of C. reinhardtii ferredoxin, which PCC 7119 proteins (Morales et al. 2000). Given includes Asp25, Glu28 and Glu29, is also likely to the likely similarities between ferredoxin binding be involved in interactions with C. reinhardtii by FNR and by Fd-glutamate synthase (Knaff Fd-glutamate synthase (Garcıá-Sańchez et al. 1996), it may well be that hydrophobic interactions 2000). The corresponding region on spinach fer- and the release of oriented water molecules from redoxin, which includes amino acid residues 26–30, the protein surfaces make substantial contribu- has been implicated in the interaction with spinach tions to stabilizing the complex between ferredoxin Fd-glutamate synthase, as has the acidic region on and Fd-glutamate synthase. spinach ferredoxin that includes residues 65–70 Less is known about the possible location of (Hirasawa et al. 1986). ferredoxin-binding domain(s) on Fd-glutamate Although the observations summarized above synthase than is known about regions on ferredoxin provide strong support for the involvement of that may be involved in binding to Fd-glutamate electrostatic interactions in stabilizing the complex synthase, although it seems likely that a ferredoxin- formed between ferredoxin and Fd-glutamate binding site on Fd-glutamate synthase lies in the synthase, the actual situation may prove to be region that contains the [3Fe-4S] cluster and FMN more complex. A very large body of evidence, prosthetic groups. It has been proposed that a loop involving the same sort of data described above for in this region (which encompasses amino acid resi- Fd-glutamate synthase, had been available sup- dues 907–933 of the Synechocystis sp. PCC 6803 porting the role of electrostatic interactions in Fd-glutamate synthase) may be part of (or close to) stabilizing the 1:1 complex formed between ferre- the site of interaction between Fd and Fd-glutamate doxin and FNR (Knaff 1996). When crystal synthase (van den Heuvel et al. 2003) and the fact structures became available recently for two fer- that this region is conserved in all Fd-glutamate redoxin/FNR complexes, one between the maize synthases is consistent with this proposal. Work leaf proteins (Kurisu et al. 2001) and one between on the X-ray crystal structure of spinach Fd- the proteins from the cyanobacterium Anabaena glutamate synthase (J.P. Allen, A. Artigas Camara, sp. PCC7119 (Morales et al. 2000), it became clear M. Hirasawa, D.B. Knaff, unpublished observa- that, although the interactions between the two tions) has not yet progressed to the point where it proteins do involve some ion pairs and hydrogen can be stated unambiguously that a similar loop is bonds, a significant number of contacts between present in the spinach enzyme and the large size of hydrophobic side chains are also involved. In fact, spinach Fd-glutamate synthase has made it impos- earlier studies of the effects of ionic strength on the sible to use peptide-mapping techniques, of the type kinetics of electron transfer from Anabaena sp. used successfully with FNR (Jelesarov et al. 1993) PCC 7120 ferredoxin to Anabaena sp. PCC 7119 and ferredoxin-dependent nitrite reductase (Dose FNR had indicated that hydrophobic, as well as et al. 1997), to identify specific lysine and arginine electrostatic, effects were likely to play an impor- residues that may be involved in binding ferredoxin. tant role in this system (Hurley et al. 1996). A Evidence from chemical modification studies microcalorimetry study of the complex formed suggests that a tryptophan residue may be present between spinach ferredoxin and spinach FNR at the ferredoxin-binding site on spinach Fd-glu- indicated that DH, the change in enthalpy associ- tamate synthase. Treatment of spinach Fd-gluta- ated with complex formation, is essentially zero mate synthase with the tryptophan-modifying and that the large, favorable negative DG (the free reagent N-bromosuccinimide resulted in equal energy change) for complex formation results losses of ferredoxin-dependent and methyl violo- entirely from the favorable positive DS (the gen-dependent activities. The time course observed entropy change) associated with complex forma- for this loss of activity was similar to that observed tion (Jelesarov and Bosshard 1994). The favorable for the modification of tryptophan residues and positive DS appears to arise from the transfer of both the activity loss and the tryptophan modifi- 203 cation were eliminated if complex formation with redoxin-binding site on spinach Fd-glutamate ferredoxin preceded treatment with N-bromosuc- synthase resembles the ferredoxin-binding site on cinimide (Hirasawa et al. 1998). These results have FNR and on nitrite reductase (Hirasawa et al. been interpreted in terms of the presence of at least 1989, 1991) and thus it seemed reasonable to one tryptophan residue, located at or near the expect that the stoichiometry of the ferredoxin/ ferredoxin-binding site of the enzyme, that plays a Fd-glutamate synthase complex would also be 1:1. role in electron transfer per se rather than in fer- However, while there is evidence from cross-link- redoxin binding (Hirasawa et al. 1998). As pro- ing (Schmitz et al. 1996), small angle X-ray scat- posals for specific roles for aromatic amino acids tering (van den Heuvel et al. 2003) and mass in electron transfer reactions between ferredoxin spectrometry (van den Heuvel et al. 2004) experi- and target enzymes exist (see for example Hurley ments supporting a 1:1 stoichiometry for the et al. 1993), one might imagine that a series of Synechocystis sp. PCC 6803 ferredoxin/Fd-gluta- properly oriented aromatic amino acids could mate synthase complex, other cross-linking bridge the space separating the electron-donating experiments support a stoichiometry of two [2Fe-2S] cluster of ferredoxin and the electron- ferredoxins:one Fd-glutamate synthase for the accepting group on Fd-glutamate synthase. While complex formed by the spinach proteins such an arrangement may perhaps exist, it should (Hirasawa et al. 1991) and the proteins from the be pointed out that at present there is no evidence green algae C. reinhardtii (Garcıá-Sańchez et al. for any special requirement for an aromatic fer- 2000) and Monoraphidium braunii (Vigara et al. redoxin amino acid in its reactions with Fd-glu- 1996). Membrane ultrafiltration experiments with tamate synthase. In fact, site-directed replacement the spinach proteins are also more consistent with of Phe65, a highly conserved ferredoxin amino a 2:1 ferredoxin:Fd-glutamate synthase stoichi- acid that had been implicated in some ferredoxin- ometry for the complex (Hirasawa et al. 1989). As dependent electron transfer reactions (Hurley the stoichiometry of the complex has important et al. 1993), by the non-aromatic amino acids implications for the enzyme mechanism (i.e., in alanine or isoleucine had only relatively modest deciding whether the two electrons required for the effects on the ability of ferredoxin from vegetative reaction arrive separately in two sequential elec- cells of the cyanobacterium Anabaena sp. PCC tron transfers from reduced ferredoxin or whether 7120 to serve as an electron donor to spinach it may be possible that a concerted transfer of two Fd-glutamate synthase (Hirasawa et al. 1998). electrons from two simultaneously bound ferre- Similar results were obtained with Fd-glutamate doxins might occur), it is of considerable impor- synthase from Synechocystis sp. PCC 6803 and tance to remove uncertainties as to the correct F65 variants of the Anabaena sp. PCC 7120 fer- value. redoxin or F63 variants of Synechocystis sp. PCC Formation of the spinach ferredoxin/spinach 6803 ferredoxin (the corresponding phenylalanine Fd-glutamate synthase complex is accompanied by residue in this ferredoxin) in which phenylalanine changes in the circular dichroism (CD) spectra of was replaced by a non-aromatic amino acid one or both proteins. These spectral perturbations (Schmitz et al. 1996). have been interpreted as arising from conforma- In the case of the ferredoxin complex with tional changes in one or both proteins that result FNR (reviewed in Knaff 1996), the higher plant from complex formation (Hirasawa et al. 1989). ferredoxin-dependent nitrite (Mikami and Ida Changes in CD spectra have also been observed as 1989) and sulfite reductases (Hirasawa et al. 1987; a consequence of complex formation between fer- Akashi et al. 1999), the ferredoxin:thioredoxin redoxin and both FNR and nitrite reductase reductases from spinach (Hirasawa et al. 1988) (Knaff 1996). In the case of the ferredoxin/FNR and the cyanobacterium Synechocystis sp. PCC complex, the availability of crystal structures for 6803 (Glauser et al. 2004), and the ferredoxin- the complex between the maize chloroplast pro- dependent nitrate reductase from the cyanobacte- teins (Kurisu et al. 2001) and the Anabaena sp. rium Synechococcus sp. PCC 7942 (Hirasawa et al. PCC 7119 proteins (Morales et al. 2000) has pro- 2004) the ferredoxin/enzyme stoichiometry of the vided documentation that protein/protein inter- complexes has been shown to be 1:1. Immuno- actions within the complex do indeed cause logical experiments support the idea that the fer- significant changes in conformation of both fer- 204 redoxin and FNR. The structures of these two tion of light and carbon signals combine to affect ferredoxin/FNR complexes have also provided expression of several genes involved in nitrogen specific information about exactly which amino assimilation (Thum et al. 2003). In higher plants, acids undergo changes in conformation and about Fd-glutamate synthase and NADH-glutamate the possible implications of these conformational synthase genes are regulated by light, and by changes for understanding the mechanism of the carbon and nitrogen metabolites. Exposure of FNR-catalyzed reaction (Morales et al. 2000; dark-grown Arabidopsis seedlings to white light Kurisu et al. 2001). Evidence obtained from increases the level of GLU1 Fd-glutamate synthase structural (van den Heuvel et al. 2003) and bio- mRNA two-fold in leaves after 72 h, while GLU2 chemical (Ravasio et al. 2002) studies on the mRNA remains constant at low levels (Suzuki and Synechocystis sp. PCC 6803 Fd-glutamate syn- Rothstein 1997; Coschigano et al. 1998). Pro- thase suggests that conformational changes asso- moter-GUS expression studies revealed that the ciated with ferredoxin binding and/or changes of GLU1 promoter of Arabidopsis Fd-glutamate the redox state of ferredoxin may be essential to synthase induces GUS reporter gene expression activate the enzyme for catalysis. after exposure of the dark-grown transgenic Although the studies described above provide a tobacco seedlings to red light (Ziegler et al. 2003). preliminary picture of some aspects of the site(s) Red light typically enhances the levels of GLU1 involved in the interaction between ferredoxin and mRNA in dark-grown Arabidopsis leaves, and Fd-glutamate synthase and of the possibility of mRNA induction is reversibly suppressed by significant conformational changes induced by subsequent exposure to far-red light in a typical protein/protein complex formation, many details phytochrome-mediated response. In contrast, of the interaction remain to be elucidated. It is to phytochrome-treatments do not affect the levels of be hoped that current attempts to obtain diffrac- GLU2 mRNA in Arabidopsis leaves (unpublished tion-quality crystals of the ferredoxin complexes data). GUS expression is induced by sucrose in the of Synechocystis sp. PCC 6803 Fd-glutamate dark albeit to a lower extent than the increase synthase (van den Heuvel et al. 2003) and of produced by red light or white light, ranging spinach Fd-glutamate synthase (J.P. Allen, A. between 2.5- and 3.5-fold (Ziegler et al. 2003). The Artigas Camara, M. Hirasawa, D.B. Knaff, accumulation of the corresponding GLU1 mRNA unpublished observations) will eventually lead to produced by sucrose addition in the dark is also three-dimensional structures that will settle the lower than that produced by light in Arabidopsis question of ferredoxin/enzyme stoichiometry (Coschigano et al. 1998; unpublished results). within the complex, will identify the interaction Sucrose also mimics the phytochrome effects by domains involved in complex formation between inducing high levels of GLN2 mRNA (16–17- fold ferredoxin and Fd-glutamate synthase and will increases) in Arabidopsis or repressing ASN1 identify specific conformational changes that arise mRNA in maize (Lam et al. 1994; Chevalier et al. from the interactions between the proteins. 1996; Oliveira and Coruzzi 1999; Thum et al. 2003). Light or sucrose induces a two- to three- fold increase in GLN1 mRNA expression in Ara- Regulation of glutamate synthases in higher plants bidopsis leaves (Oliveira and Coruzzi 1999). However, supplying sucrose or glucose to excised Light at low fluency is perceived at specific dark-grown barley leaves does not induce wavelengths by the photoreceptors and signal Fd-glutamate synthase mRNA or produce transduction then triggers chloroplast differentia- detectable enzyme activity (Pajuelo et al. 1997). In tion, circadian rhythms and a number of physio- etiolated maize leaves, Fd-glutamate synthase logical and molecular responses in plants (Neff mRNA increases as early as 6 h after the onset of et al. 2000). Studies of gene expression in mutants illumination, and it accumulates four-fold, reach- suggest that plants have sensing and signal trans- ing a level equivalent to that found in green leaves duction mechanisms that respond to the cellular (Sakakibara et al. 1992a). Induction of GLU concentrations of carbon and nitrogen (sucrose, mRNA in maize leaves is at least in part mediated ) + NO3 ,NH4 , glutamine, glutamate, 2-oxogluta- by phytochrome (Suzuki et al. 2001). Fd-glutarate, etc.) (Coruzzi and Zhou 2001). The interac- mate synthase mRNA accumulation produced by 205 the light correlates with de novo synthesis of the The control of glutamate synthase expression by ) ) enzyme protein (Sakakibara et al. 1992b; Pajuelo NO3 and the downstream products of NO3 et al. 1997; Suzuki et al. 2001). Induction of assimilation has also been extensively investigated. Fd-glutamate synthase by a phytochrome-medi- In the leaves of detached maize seedlings, a supply of ) ated pathway is reflected in an increased ratio of NO3 results in increases in the mRNA levels of glutamate to glutamine, which is close to the value both Fd-glutamate synthase (l.5-fold) and NADH- observed in leaves exposed to white light (Suzuki glutamate synthase (2.5-fold) within 4 h (Sakaki- et al. 2001). In contrast, light has no effect on bara et al. 1997). In these plants, the kinetics NADH-glutamate synthase levels in leaves observed for the increases in NAR, NII and GS2 (Suzuki and Rothstein 1997; Turano and Muhitch mRNA levels are all quite similar (Sakakibara et al. 1999; Suzuki et al. 2001). As a result of these 1997). Three- to five fold increases in Fd-glutamate differential light effects, Fd-glutamate synthase synthase mRNA have also been observed in illu- ) accounts for more than 95% of the total gluta- minated etiolated maize leaves in response to NO3 + ) mate synthase activity in photosynthetic plant or NH4 . In the absence of light, both NO3 and + tissues, and NADH-glutamate synthase accounts NH4 induce Fd-glutamate synthase gene expres- for only a small residual activity. The induction of sion to much lower levels (Suzuki et al. 1996; Fd-glutamate synthase mRNA via phytochrome Pajuelo et al. 1997). A similar time course has been signal transduction may include blue/UV-A light observed for labeling of the Fd-glutamate synthase receptors in Scot pine (Elminger and Mohr 1991) with [35S] methionine. Synthesis of the enzyme and in Spirodela polyrhiza (Teller et al. 1996). protein is blocked by cycloheximide (Suzuki et al. However, Pinus pinaster, another pine species, 1996). Nitrate induction of Fd-glutamate synthase accumulates mRNA and protein for Fd-glutamate mRNA is not affected by the phosphatase inhibitors synthase, as well as mRNA for the photosynthetic okadaic acid and calyculin A in maize leaves, while genes of Rubisco large and small subunits and the inhibitors repress the mRNA levels of NAR, NII chlorophyll a/b binding proteins in a light-inde- and GS2 (16–69%) (Sakakibara et al. 1997). The pendent manner (Garcıá-Gutie´rrez et al. 1995). In protein kinase inhibitors W-7 and W-5 slightly addition to the phytochrome-mediated induction, reduce the nitrate-dependent accumulation of Fd-glutamate synthase mRNA expression in green Fd-glutamate synthase mRNA (8–29%), implying leaves changes diurnally, with induction early in a possible involvement of calmodulin-regulated the morning and slight repression at the middle of process or Ca2+-dependent but calmodulin-inde- the day (Suzuki et al. 2001; Ferrario-Me´ry et al. pendent protein kinases in the nitrate-signal trans- 2002). Both the GLU1 promoter and GLU1 duction pathway for regulation of GLU expression mRNA are up-regulated by light and sucrose, (Sakakibara et al. 1997). Nitrate also induces suggesting that GLU1-encoded Fd-glutamate mRNA for Fd-glutamate synthase, GS1 and GS2 in ) synthase functions under conditions of high tobacco leaves 4 h after exposure to NO3 (Scheible sucrose availability produced by the photosyn- et al. 1997). Multiple nitrogen metabolites partially thesis. Transcript levels for nitrate reductase counteract the low mRNA levels produced by ) (NAR) and nitrite reductase (NII) are also high at nitrogen starvation, i.e., NO3 or glutamine induces the end of the night and decreased during the light GS2 mRNA and Fd-glutamate synthase mRNA, + period (Scheible et al. 2000). Several lines of evi- and NH4 increases Fd-glutamate synthase dence indicate that the light- and circadian- mRNA. A specific effect of these nitrogen com- responsive elements provide the binding sites for pounds can thus be excluded (Migge and Becker transcription factors, which are diurnally induced 1996). However, in suspensions of cultured rice cells ) (Borello et al. 1993; Anderson et al. 1994; Staigner deprived of nitrogen, NO3 induced Fd-glutamate and Apel 1998). Both phytochromes and crypto- synthase mRNA levels whereas no induction is + chromes are involved in maintaining the rhythm produced when NH4 or the amino acids gluta- close to 25 h under red and blue light in Arabid- mine, glutamate, asparagine or aspartate are sup- opsis (Millar et al. 1995; Somers et al. 1998). Also plied (Watanabe et al. 1996; Hirose and Yamaya mRNA is post-transcriptionally light-regulated by 1999). In nitrogen-starved rice leaves, neither coupling to photosynthetic electron transport at Fd-glutamate synthase nor NADH-glutamate syn- + the level of mRNA stability (Petracek et al. 1998). thase are affected by the application of NH4 206

(Yamaya et al. 1995). Arabidopsis plants starved for PII-like protein present in chloroplasts serves as a ) + nitrogen and then exposed to NO3 or NH4 - sensor of glutamine by changing uridylylation and treatment were studied using microarrays repre- adenylation states or is involved in glutamine senting about 21,000 genes (Meyer et al. 2003). The metabolisms in Arabidopsis (Hsieh et al. 1998; Jiang ) addition of NO3 induced mRNA levels for GLU1 and Ninfa 1999). and GLT in leaves (Figure 2). The addition of In roots, the levels of Fd-glutamate synthase + NH4 only slightly affected the mRNA levels for mRNA increases rapidly, showing a 6.5-fold ) ) GLU1, GLU2 and GLT. In contrast, neither NO3 increase within 30 min after exposure to NO3 , + nor NH4 affects Fd-glutamate synthase gene while the mRNA levels for NAR and GS2 peak expression after exposure of etiolated soybean after 4 h (Redinbaugh and Campbell 1993). A leaves to light (Turano and Muhitch 1999). The lack transient four-fold accumulation of Fd-glutamate ) of induction by NO3 is also observed for Fd-glu- synthase mRNA is produced in etiolated coleop- ) tamate synthase and GS2 genes in maize leaves tiles of rice only 30 min after addition of NO3 , (Redinbaugh and Campbell 1993). The decrease in synchronous with the labeling of Fd-glutamate Fd-glutamate synthase mRNA at the middle of the synthase peptide with [35S]methionine (Mattana day is reminiscent of a repression of some of nitro- et al. 1996). In rice roots, NADH-glutamate syn- gen assimilatory genes during the light period thase mRNA is detected in sclerenchyma cells in + (Scheible et al. 2000; Ferrario-Me´ry et al. 2002). the inner cell-layer as early as 3–6 h after NH4 is ) Following the diurnal changes in the levels of NO3 supplied and the level subsequently declined + and NH4 (Scheible et al. 2000; Matt et al. 2001; (Ishiyama et al. 1998, 2003). In contrast, little Ferrario-Me´ry et al. 2002; Stitt et al. 2002), gluta- change is observed in the levels of Fd-glutamate mine and 2-oxoglutatate (2-OG) may regulate the synthase and GS isoproteins in roots (Yamaya + NH4 assimilation by acting antagonistically. It et al. 1995). This transient expression correlates has been suggested that an allosteric effector of the with the appearance of GUS activity in the

NO3- 3000 2500 GLU1 Fd-GOGAT 2000 GLU2 Fd-GOGAT 1500 GLT1 NADH-GOGAT 1000

Expression level 500 0 N N NO3- NO3- starvation starvation induction induction Leaf Root Leaf Root

NH4+ 3000 2500 GLU1 Fd-GOGAT 2000 GLU2 Fd-GOGAT 1500 GLT1 NADH-GOGAT 1000 Expression level 500 0 N N srarvation NH4+ NH4+ starvation Root induction induction Leaf Leaf Root Figure 2. Histograms of the expression of genes for ferredoxin-glutamate synthase (GLU1 and GLU2) and NADH-glutamate synthase (GLT) in leaves and roots of Arabidopsis thaliana. Thirty-five-day-old Arabidopsis thaliana plants were subjected to nitrogen starvation ) + for 10 days, and nitrogen was then supplied in the form of 10 mM NO3 or 5 mM NH4 for 6h. Using high-density oligonucleotide probe microarray (Affymetrix GeneChip) analysis (Meyer et al. 2003), expression of 21 000 genes was examined in the leaves and roots. Nitrate induced 1.6- and 3.7-fold increases in GLU1 mRNA and GLT mRNA, respectively in leaves, and a 4.4-fold-increase in GLT mRNA in roots. Ammonium induced l.4- and 4.5-fold increases in GLU2 mRNA and GLT mRNA, respectively in roots. 207 sclerenchyma cells of transgenic plants containing Mutants and transgenic plants affected in glutamate a NADH-glutamate synthase GLT promoter-GUS synthase reveal the roles of the enzyme in nitrogen + fusion 3 h after NH4 treatment (Ishiyama et al. metabolism in higher plants 2003). In the absence of cycloheximide, NADH- glutamate synthase is detected later in the outer In most tropical and subtropical plants, primary cell-layers including epidermal and exodermal cells nitrate assimilation into amino acids occurs pre- (Ishiyama et al. 1998, 2003). In nitrogen-starved dominantly in leaf chloroplasts. In some plant ) Arabidopsis,NO3 induced GLT mRNA for species, such as temperate legumes and maize, NADH-glutamate synthase in roots (Figure 2). efficient nitrate assimilation also occurs in root + The addition of NH4 also induced GLT mRNA plastids (Pate 1980). In addition to primary nitrate + in roots, while GLU1 mRNA was barely detected reduction, NH4 is released within mitochondria (Figure 2). Ammonium and glutamine act as of mesophyll cells in C3 plants at rates 5–10-fold metabolic signals for the induction of the NADH- higher than rates of nitrate reduction (Somerville + glutamate synthase gene, while other amino acids, and Ogren 1980). As NH4 assimilation into ami- i.e. glutamate, asparagine, aspartate, alanine or no acids via glutamate synthase is coupled to the serine, have little effect in rice roots (Ishiyama GS-catalyzed reaction, the physiological role of et al. 1998). Okadaic acid caused the continuous Fd-glutamate synthase and NADH-glutamate accumulation of NADH-glutamate synthase synthase are tightly linked to GS2 and GS1, which mRNA, and an okadaic acid-sensitive reversible play tissue- and organ-specific roles (Figure 3) protein phosphorylation appears to be involved in (Lam et al. 1996). The in vivo function of different the signal transduction pathway (Ishiyama et al. forms of GS and glutamate synthase has been 1998; Hirose and Yamaya 1999). investigated by the characterization of mutants and

MesophyllLeaf / shoot Vasculature Seed Glutamate 2-Oxo-glutarate Glutamate 2-Oxo-glutarate Storage Fd-GOGAT Fd-GOGAT / NADH-GOGAT proteins Asparagine Asparagine Glutamate Glutamine Glutamate Glutamine

GS2 NH + NH + GS1 Vacuole 4 4 Amino acids - NO3 Photorespiration - Amino acids NO3 Transport

Xylem Phloem Transport

Amino acids Vacuole Glutamate 2-Oxo-glutarate NO - 3 NADH-GOGAT / Fd-GOGAT Asparagine Glutamate Glutamine

Storage GS1

- + NO3 NH4 Root

- + NO3 NH4 Figure 3. A model for the role of Fd-glutamate synthase and NADH-glutamate synthase in nitrogen assimilation and nitrogen transport. Plants import nitrate and ammonium ion into the roots when available in the soil. Nitrate storage in the vacuoles and nitrate reduction to nitrite and, subsequently into ammonium occur in the roots and in the leaves. Ammonium ion issued from different pathways is assimilated into glutamine and glutamate by the sequential reaction of glutamine synthetase (cytosolic GSI and chloroplastic GS2) and glutamate synthase (Fd-glutamate synthase and NADH-glutamate synthase). Glutamine and glutamate are used for amino acid, nucleic acid and protein biosynthesis. Amino acids are in part mobilized into the xylem for transport to the shoots or into the phloem for transport to other organs. 208 transgenic plants. Photorespiratory mutants et al. 2000), and the strict correlation between the defective in Fd-glutamate synthase and/or GS are mutant phenotype and enzyme deficiency provides isolated by conditional lethal phenotype screenings evidence that the GS/Fd-glutamate synthase cycle in Arabidopsis and barley (Somerville and Ogren plays the indispensable role for the reassimilation + 1980; Kendall et al. 1986; Blackwell et al. 1988). of photorespiratory NH4 .InArabidopsis, the loss The Arabidopsis gls mutants contain 2–4% of the of the GLU1 isoform of Fd-glutamate synthase wild-type Fd-glutamate synthase activity in green cannot be compensated for by the second GLU2 leaves, and NADH- or NADPH-dependent reac- isoform of Fd-glutamate synthase or by NADH- tions account for 4% of the total glutamate syn- glutamate synthase, both of which are constitutive thase activity in leaves (Somerville and Ogren and present at low levels in leaves. Promoter-GUS 1980). There are two expressed genes GLU1 and fusion studies with transgenic tobacco plants GLU2 for Fd-glutamate synthase, and a single GLT demonstrated that the Arabidopsis GLU1 promoter for NADH-glutamate synthase in Arabidopsis directs expression of the uidA reporter gene in the (Figure 1). GLU1 mRNA for the major Fd-g1u- chloroplasts of palisade and spongy parenchyma of tamate synthase is primarily expressed in green the mesophyll (Ziegler et al. 2003; unpublished leaves, similar to GLN2 gene for chloroplastic GS2 results), the major site for photorespiration. Im- that is highly expressed in leaves (Peterman and munolocalization studies detected Fd-glutamate Goodman 1991; Suzuki and Rothstein 1997; synthase protein in the chloroplast stroma of Coschigano et al. 1998; Lancien et al. 2002). Pre- mesophyll in tomato (Botella et al. 1988). These sumably, the lethal chlorosis is caused by the results provide evidence for the efficient in vivo mutation of highly expressed GLU1 gene, which is function of Fd-glutamate synthase in photorespi- mapped at the same region as a gls allele on chro- ratory nitrogen cycling. In the absence of the major mosome 5 while the GLU2 gene on chromosome 2 Fd-glutamate synthase in barley mutants (0.5– is not affected (Coschigano et al. 1998). The Ara- 1.3% of the wild-type leaf activity), the impairment + bidopsis gls mutants typically have decreased levels of reassimilation of photorespiratory NH4 leads + of GLU1 mRNA and the corresponding low Fd- to the accumulation of NH4 , even though glutamate synthase activity in leaves (1.7%) and NADH-glutamate synthase remains at the wild- roots (25%) (Somerville and Ogren 1980; Suzuki type level (2.2–3.2% of the wild-type Fd-glutamate and Rothstein 1997). The photorespiratory mutant synthase activity) (Blackwell et al. 1988). In addi- phenotype has also been demonstrated in trans- tion to the decrease in glutamate, the rate of CO2 genic tobacco lines expressing a 936-bp partial Fd- fixation declines to 25–30% of the initial rate at glutamate synthase cDNA in the antisense orien- high light intensity (Blackwell et al. 1988). The tation (Ferrario-Me´ry et al. 2000). These trans- inhibition of photosynthetic CO2 fixation in the genic tobacco plants exhibit Fd-glutamate synthase chlorotic plants might be caused by the inhibition activity that has been decreased by 10–81% in of CO2 exchange arising from the inactivation of leaves and 17–65% in roots in three transgenic CO2 assimilating enzymes, the lack of amino + lines. Following a shift from CO2-enriched air donors or by high concentrations of NH4 , which + (0.4%) to ambient air for 48 h, NH4 accumulates uncouples photophosphorylation. in amounts as high as 50–300 nmol)1 mg)1 leaf dry Analysis of Fd-glutamate synthase mutants 14 weight in a manner that increases in proportion to pulsed with CO2 after a short period of photo- the inhibition of Fd-glutamate synthase activity. synthesis in ambient air revealed that the mutants The accumulation of glutamine and 2-OG corre- are affected in the levels of organic acids, sugar + sponds approximately to the increase in NH4 in phosphates and amino acids within 20 min. The + these plants. This is expected because NH4 must mutants partially recover the ability to fix CO2 and + be refixed by GS and 2-OG is the substrate for assimilate NH4 during subsequent exposure to Fd-glutamate synthase (Ferrario-Me´ry et al. 2000). non-photorespiratory conditions, i.e., either in Following the initial decrease, the levels of gluta- high CO2 or in darkness (Somerville and Ogren mate stabilize as further synthesis by Fd-glutamate 1980; Kendall et al. 1986). In high CO2, Arabid- synthase is prevented. The decrease in Fd-gluta- opsis gls mutants exhibit wild-type levels of total mate synthase activity by even 20% in leaves limits protein and of chlorophyll (Grumbles 1989). In the + photorespiratory NH4 cycling (Ferrario-Me´ry dark, barley Fd-glutamate synthase mutants, 209 which exhibit Fd-glutamate synthase activity at a during the dark to light transition in the antisense level only 66% of that characteristic of control transgenic plants. In support of a role of the GLU1- plants, maintain glutamate contents and gluta- encoded Fd-glutamate synthase in primary nitro- mate/glutamine ratios close to those of the wild- gen assimilation, genetic studies showed that a gls1 type plants (Ha¨ usler et al. 1994). This implies that mutant is impaired in primary nitrogen assimila- the GS/glutamate synthase cycle functions in vivo tion: unlike the case of wild-type Arabidopsis, the in the dark, while in Arabidopsis Fd-glutamate gls1 mutant (NA60) was unable to respond to ) synthase mutants, the impairment of nitrogen increasing concentrations of exogenous NO3 and + assimilation results in an increase in glutamine/ NH4 in the increase of chlorophyll accumulation glutamate ratios relative to those typical of the when photorespiration was suppressed in high CO2 wild-type plants (Lancien et al. 2002). Analysis of (Coschigano et al. 1998). However, this does not the kinetics of labeling with 15N revealed that the eliminate the possibility that the residual NADH- labeling of [5-15N]glutamine, [2-15N]glutamate, glutamate synthase in the gls1 mutant plays a [2-15N]glutamine and double labeling of physiological function in the leaf primary nitrogen [2,5-15N]glutamine occurs by the sequential reac- assimilation in place of GLU1-encoded Fd-gluta- tions of GS and glutamate synthase, albeit at lower mate synthase. Similarly, it is not clear whether the rates in the dark than in the light in the leaves of second GLU2 isoform of Fd-glutamate synthase is + some plant species. These studies support the view involved in the leaf primary NH4 assimilation, or that the GS/glutamate synthase cycle can operate whether this enzyme is responsible for supplying in the dark, even in the absence of energy gener- basal levels of glutamate for protein synthesis in ated by photosynthesis. Further evidence for leaves. + NH4 assimilation in the dark was provided by In higher plants, including Fd-glutamate syn- experiments utilizing antisense Fd-glutamate synthase mutants and antisense Fd-glutamate synthase + thase tobacco plants, in which NH4 assimilation transgenic plants, Fd-glutamate synthase mRNA is occurs with the concomitant decrease in glutamine expressed at high levels in leaves and low levels in and 2-OG and constant level of glutamate during roots. In contrast, NADH-glutamate synthase gene the dark phase of dark to light transitions expression predominates in roots, along with a (Ferrario-Me´ry et al. 2002). The 15N labeling and concomitant higher expresssion of the GLN1 genes inhibitor studies on the NADH-GDH null encoding cytosolic GS1 (Peterman and Goodman mutants suggest that NADH-GDH does not act in 1991; Suzuki and Rothstein 1997; Coschigano et al. the assimilatory direction (Magalha˜ es et al. 1990; 1998). These organ-specific expression patterns of Aubert et al. 2001). Therefore, these results imply the distinct glutamate synthase genes imply a spe- + that NH4 is assimilated in the reaction of GS via cific role of the Fd-glutamate synthase isoforms and a residual Fd-glutamate synthase and/or NADH- NADH-glutamate synthase. When 15N-nitrate was glutamate synthase in the dark, although at lower supplied to barley Fd-glutamate synthase mutants rates than in the light. in high CO2, there was little difference in the amino The observation that the Fd-glutamate synthase acid levels in roots compared with the wild-type mutants and antisense Fd-glutamate synthase plants, other than a three-fold increase in glutamine transgenic plants are fully viable when photores- and one-third decrease in glutamate (Joy et al. piration is suppressed in high CO2 suggests that GS 1992). It thus appears that the major part of labeled and a low Fd-glutamate synthase and/or NADH- nitrogen is transported as glutamine (86%) in the glutamate synthase remaining in the affected plants xylem to leaves (Joy et al. 1992). In spite of the high can provide sufficient amino acids during primary glutamine/glutamate ratio, which is indicative of the ) NO3 reduction, which occurs primarily in leaves. lower levels of glutamate synthase, both Fd-gluta- The antisense Fd-glutamate synthase transgenic mate synthase and NADH-glutamate synthase tobacco plants lack NADH-glutamate synthase activities in roots remain at wild-type levels activity in leaves (Ferrario-Me´ry et al. 2000). accounting for 3% and 4% of the leaf Fd-glutamate Consequently, the residual Fd-glutamate synthase synthase activity, respectively (Joy et al. 1992). + is expected to be involved in primary NH4 Using a promoter-GUS transgene, rice NADH- assimilation in the leaf mesophyll cells, and the GS/ glutamate synthase promoter expression was glutamate synthase cycle operates at least partly localized in the vascular bundles of the developing 210 leaf blades and in the dorsal and lateral bundles of dant role with NADH-glutamate synthase in developing grains (Kojima et al. 2000). This pro- nitrogen translocation in the antisense Fd-gluta- moter expression pattern overlaps with the location mate synthase transgenic tobacco plants. Histo- of immuno-detected NADH-glutamate synthase chemical studies, which revealed that the protein (Hayakawa et al. 1994; Ishiyama et al. Arabidopsis GLU1 promoter for Fd-glutamate 2003). Because NADH-glutamate synthase and synthase directs GUS reporter gene expression in cytosolic GS1 genes share a similar organ-specific the vascular cells and in the root meristems of expression pattern, it has been proposed that the transgenic tobacco plants, support this hypothesis GS1/NADH-glutamate synthase cycle supplies the (Ziegler et al. 2003, unpublished data). This obser- amino acids for nitrogen transport from roots to vation reinforces the view that the GS/Fd-gluta- leaves or from sources to sinks through the vascular mate synthase cycle controls the assimilation of tissues (Edwards et al. 1990; Carvalho et al. 2000; nitrogen that is subsequently transported, with Tobin and Yamaya 2001). Moreover, a role of amino acids serving as the nitrogen carriers. The 15 + 15 NADH-glutamate synthase in nitrogen transport kinetics of NH4 labeling into [5- N]glutamine, was proposed using an Arabidopsis glt-T knock-out [2-15N]glutamate, [2-15N]glutamine and [2,5-15N]- mutant which lacks NADH-glutamate synthase glutamine correlate with the operation of GS and mRNA and activity, while the activity of GLU1 glutamate synthase, not only in sink leaves but also isoform of Fd-glutamate synthase remains at the in source leaves of tobacco (unpublished results). wild-type level in leaves (Lancien et al. 2002). The These data imply that GS and even a low amount of glt-T mutant exhibits a deleterious growth pheno- Fd-glutamate synthase and/or NADH-glutamate type, and fresh weight and chlorophyll contents are synthase located in the mesophyll and vascular cells reduced by 20% and 30%, respectively, in the air- provide sufficient glutamine and glutamate for grown leaves. The mutant contains a 2.2-fold higher transport to maintain the partitioning of nitrogen level of glutamine, and glutamate and aspartate between leaves and roots. In contrast to the barley decreased by two-thirds relative to the wild-type Fd-glutamate synthase mutants, which have wild- levels. These effects have been interpreted as arising type levels of root NADH-glutamate synthase from the impairment of glutamate synthesis in leaf activity, NADH-glutamate synthase is missing in veins caused by NADH-glutamate synthase defi- the roots of Arabidopsis glt-T knock-out mutant ciency (Lancien et al. 2002). Therefore, it is and in the roots of antisense Fd-glutamate synthase hypothesized that Fd-glutamate synthase does not transgenic tobacco plants (Ferrario-Me´ry et al. compensate for NADH-glutamate synthase in its 2000; Lancien et al. 2002). It is possible that, instead role in nitrogen translocation in the vascular tissues of NADH-glutamate synthase supporting the (Lancien et al. 2002). A substantial portion of the translocation of amino acids, glutamine and gluta- nitrogen arriving at the leaf veins through the xylem mate are formed in the roots by GLU2-encoded is stored, metabolized and redistributed through the Fd-glutamate synthase isoform, which is preferen- phloem sieve tube elements to heterotrophic sink tially expressed in the roots (Lancien et al. 2002) or tissues (Andrew 1986; Pate and Layzell 1990). In by Fd-glutamate synthase, which is slightly addition, 14C- and 15N-labeling experiments enhanced in the roots of the antisense Fd-glutamate showed that the bulk of nitrogen in the leaves is synthase transgenic tobacco under elevated CO2 transported to roots, and a portion of the reduced (Ferrario-Me´ry et al. 2000). Glutamate can also be nitrogen in roots is in turn transported to shoots as provided by transaminase reactions. In spite of glutamine, asparagine, glutamate and aspartate differences in the availability of the energy between (Cooper and Clarkson 1989; Jeschke and Pate the roots and photosynthetic leaves, NADPH can 1991). In spite of the essential role of NADH-glu- serve as a source of electrons for the Fd-glutimate tamate synthase for amino acid cycling in vascular synthase reaction by reducing a root specific Fd tissues, the antisense Fd-glutamate synthase trans- isoform in a reaction catalyzed by a root specific genic tobacco lines provide sufficient levels of glu- isoform of ferredoxin-NADP+ reductase (Suzuki tamate without NADH-glutamate synthase, which et al. 1985; Bowsher et al. 1992; Yonekura- is missing in both leaves and roots in high CO2 Sakakibara et al. 2000). In alfalfa root nodules, (Ferrario-Me´ry et al. 2000). This implies that NADH-glutamate synthase transcripts accumulate Fd-glutamate synthase presumably plays a redun- predominantly in the N2-fixing zone of infected cells 211

(Vance et al. 1995; Trepp et al. 1999a). Antisense NADPH-glutamate synthase, NADH-glutamate expression of a 2.5-kb NADH-glutamate synthase synthase and Fd-glutamate synthase. The three cDNA decreased the levels of NADH-glutamate forms are present in a wide variety of organisms synthase transcript and protein in root nodules to and exhibit both shared and unique structural 40–50% of those found in control plants. As a properties. Fd-glutamate synthase and NADH- result, the transgenic lines exhibit impairment in glutamate synthase serve to synthesize the amino + NH4 assimilation and a moderate chlorotic phe- acids within the different compartments of the notype, with pale green leaves and stems under plant cells, which are located in the photosynthetic symbiotic N2 fixation conditions (Schoenbeck et al. and non-photosynthetic organs, in tight concert 2000; Cordoba et al. 2003). Cytosolic GS1, encoded with GS and with other enzymes of nitrogen and by two genes in alfalfa root nodules, is located in the carbon metabolism. Genetic and molecular analy- transfer cells of vasculature pericycles, and it is ses using transgenic plants and mutants deficient in likely that GS1 plays a role in transporting amino Fd-glutamate synthase or NADH-glutamate syn- acids away from the infected cells of amido-trans- thase have provided an excellent tool for studying porting nodules (Carvalho et al. 2000). NADH- differences in the expression of Fd-glutamate syn- glutamate synthase protein can be immuno-chemi- thase and NADH-glutamate synthase in response cally detected in the proximal part of older alfalfa to environmental stimuli such as light, metabolic root nodules (33 days old) where N2-fixation is regulation, cell- and organ-specific control and inefficient, and it is hypothesized that the GS1/ developmental regulation. Molecular analyses and NADH-glutamate synthase pathway is involved, at biochemical and 15N labeling approaches have led least to some extent, in the nitrogen remobilization to a better understanding of the important regu- in senescing root nodules (Trepp et al. 1999b). latory mechanisms that affect ammonium assimi- Apparently, Fd-glutamate synthase cannot com- lation into glutamine and glutamate catalyzed by pensate for the reduced activity of NADH-gluta- the GS/Fd-glutamate synthase and/or GS/NADH- + mate synthase in the primary NH4 assimilation, glutamate synthase cycles and the amino acid even though the levels of Fd-glutamate synthase metabolism. Both of these GS/glutamate synthase transcript increase in the transgenic root nodules cycles occur, in both light-dependent and light- (Schoenbeck et al. 2000). This could be explained by independent versions, throughout all stages of the tissue-specific expression of Fd-glutamate syn- plant life starting from the germination and thase and NADH-glutamate synthase, although the extending through senescence. It is certainly not exact cell types specific to Fd-glutamate synthase unreasonable to hope that these basic studies in the are not defined in root nodules. Moreover, trans- nitrogen assimilation and nitrogen metabolism in formed tobacco plants over-expressing root plants, reinforced by structural and mechanistic NADH-glutamate synthase (10–40% above the studies of the enzymes involved, will ultimately wild-type levels) exhibit 10–20% increases in shoot provide tools for improving nitrogen use efficiency biomass (expressed as carbon and nitrogen con- under field conditions. ) + tents) when using NO3 or NH4 as the sole nitrogen source (Chichkova et al. 2001). This could indicate a significant contribution of NADH-glu- Acknowledgements + tamate synthase during primary NH4 assimilation in roots. The authors would like to thank Dr Masakazu Hirasawa and Dr Maria Vanoni for helpful dis- cussions. Work in DBK’s laboratory was sup- Conclusion ported by a grant from the United States Department of Energy (DE-FG02-99ER20346). Inorganic nitrogen assimilation into glutamine, glutamate, asparagine and aspartate and the amino References acid metabolism are the essential processes for plant growth and development. Biochemical and Akashi T, Matsumura T, Ideguchi T, Iwakiri K, Kawakatsu T, molecular studies have demonstrated that gluta- Taniguchi I and Hase T (1999) Comparison of the electro- mate synthase occurs in three distinct forms: static binding sites on the surface of ferredoxin for two 212

ferredoxin-dependent enzymes, ferredoxin-NADP+ reduc- rubrun grown under nitrogen-fixing conditions. Biochem J tase and sulfite reductase. J Biol Chem 41: 29399–29405 279: 151–154 Anderson SL, Teakle GR, Martino-Catt SJ and Kay SA (1994) Carvalho H, Lescure N, de Billy F, Chabaud M, Lima L, Circadian clock- and phytochrome-regulated transcription is Salema R and Cullimore J (2000) Cellular expression and conferred by a 78 bp cis-acting domain of Arabidopsis CAB2 regulation of the Medicago truncatula cytosolic glutamine promoter. Plant J 64: 457–470 synthetase genes in root nodules. Plant Mol Biol 42: 741–756 Anderson MP, Vance CP, Heichel GH and Miller SS (1989) Castan˜ oI, Flores N, Valle F, Covarrubias AA and Bolivar F Purification and characterization of NADH-glutamate syn- (1992) gltF, a member of the gltBDF operon of Escherichia thase from alfalfa root nodules. Plant Physiol 90: 351–358 coli, is involved in nitrogen-regulated gene expression. Mol Andrew M (1986) The partitioning of nitrate assimilation Microbiol 6: 2733–2741 between root and shoot of higher plants. Plant Cell Environ Castillo A, Taboada H, Mendoza A, Valderrama B, Encarna- 9: 511–519 cion S and Mora J (2000) Role of GOGAT in carbon and Aubert S, Bligny R, Douce R, Gout E, Ratcliffe RG and nitrogen partitioning in Rhizobium etli. Microbiol 146: 1627– Roberts JKM (2001) Contribution of glutamate dehydro- 1637 genase to mitochondrial glutamate metabolism studied by Chen F-L and Cullimore JV (1988) Two isoenzymes of 13C and 31P nuclear magnetic resonance. J Exp Bot 52: NADH-dependent glutamate synthase in root nodules of 37–45 Phaseolus vulgaris L. Purification, properties and activity Avila C, Mrquez AJ, Pajuelo P, Cannell ME, Wallsgrove RM changes during nodule development. Plant Physiol 179: and Ford BG (1993) Cloning and sequence analysis of a 1411–1417 cDNA for barley ferredoxin-dependent glutamate synthase Chevalier C, Bourgeois E, Just D and Raymond P (1996) and molecular analysis of photorespiratory mutants deficient Metabolic regulation of asparagine synthetase gene expres- in the enzyme. Planta 189: 475–483 sion in maize root tips. Plant J 9: 1–11 Bast E (1977) Utilization of nitrogen compounds and ammonia Chichkova S, Arellano J, Vance CP and Hernańdez G (2001) assimilation by Chromatiaceae. Arch Microbiol 113: 91–94 Transgenic tobacco plants that overexpress alfalfa NADH- Belitsky BR, Janssen PJ and Sonenshein AL (1995) Sites glutamate synthase have higher carbon and nitrogen con- required for GltC-dependent regulation of Bacillus subtilis tent. J Exp Bot 52: 2079–2087 glutamate synthase expression. J Bacteriol 177: 5686–5695 Chiu JY and Shargool PD (1979) Importance of glutamate Binda C, Bossi RT, Wakatsuki S, Arzt S, Coda A, Curti B, synthase in glutamate synthesis by soybean cell suspension Vanoni MA and Mattevi A (2000) Cross-talk and ammonia cultures. Plant Physiol 63: 409–415 channeling between active centers in the unexpected domain Cogoni C, Valenzuela L, Gonza´lez-Halphen D, Oliveira H, arrangement of glutamate synthase. Struct Fold Des 15: Macino G, Ballario P and Gonza´lez A (1995) Saccharomyces 1299–1308 cerevisiare has a single glutamate synthase gene coding for a Blackwell RD, Murray AJS, Lea PJ and Joy KW (1988) plant-like high-molecular-weight polypeptide. J Bacteriol Photorespiratory amino donors, sucrose synthesis and the 177: 797–798 induction of CO2 fixation in barley deficient in glutamine Cooper HD and Clarkson DT (1989) Cycling of amino- synthetase and/or glutamate synthase. J Exp Bot 39: 845–858 nitrogen and other nutrients between shoots and roots in Borello U, Ceccarelli E and Giuliano G (1993) Constitutive, cereals – a possible mechanism integrating shoot and root light-responsive and circadian clock-responsive factors com- in the regulation of nutrient uptake. J Exp Bot 40: 753– pete for the different Ibox elements in plant light regulated 762 promoters. Plant J 4: 611–619 Cordoba E, Shishkaava S, Vance CP and Hermańdez G (2003) Botella JR, Verbelen JP and Valpuesta V (1988) Immunocy- Antisense inhibition of NADH glutamate synthase impairs tolocalization of ferredoxin-GOGAT in the cells of green carbon/nitrogen assimilation in nodule alfalfa (Medicago leaves and cotyledons of Lycopersicon esculentum. Plant sativa L). Plant J 33: 1037–1049 Physiol 87: 255–257 Cordovilla MP, Prez J, Ligero F, Lluch C and Valpuestra V Bowsher CG, Boulton EL, Rose J, Nayagam S and Emes MJ (2000) Partial purification and characterization of NADH- (1992) Reductant for glutamate synthase is generated by the glutamate synthase from faba bean (Vicia faba) root oxidative pentose phosphate pathway in non-photosynthetic nodules. Plant Sci 150: 121–128 root plastids. Plant J 2: 893–898 Coruzzi GM and Zhou L (2001) Carbon and nitrogen sensing Brenchley JE, Baker CA and Patil LG (1975) Regulation of and signaling in plants: emerging ‘matrix effects’. Curr Opin ammonia assimilatory enzymes in Salmonella typhimurium. Plant Biol 4: 247–253 J Bacteriol 124: 182–189 Coschigano K, Melo-Oliveira R, Lim J and Coruzzi GM (1998) Bult CJ, White O, Olsen GJ, Zhou L, Fleishman RD, Sutton GG, Arabidopsis gls mutants and distinct Fd-GOGAT genes: Blake JA, Fitzgerald LM, Clayton RA, Gocayne JD, Kerla- implication for photorespiration and primary nitrogen vage AR, Doygherty BA, Tomb J-F, Adams MD, Reich CI, assimilation. Plant Cell 10: 741–752 Overbeek R, Krikness EF, Weinstoock KG, Merrick JM, Deane SM and Rawling DE (1996) Cloning and sequencing of Glodek A, Scott JL, Geoghagen NSM, Weidman JF, Fuhr- the gene for the Thiobacillus ferrooxidans ATCC33020 mann JL, Nguyen D, Utterback TR, Kelly JM, Peterson JD, glutamate synthase (GOGAT) small subunit and comple- Shadow PW, Hanna MC, Cotton MD, Roberts KM, Hurst mentation of an Escherichia coli gltD mutant. Gene 177: MA, Kaine BP, Borodovsky M, Klein H-P, Fraser CM, Smith 261–263 HO, Woese CR and Venter JC (1996) Complete genome Dincturk HB and Knaff DB (2000) The evolution of glutamate sequence of the Methanogenic archaean, Methanococcus synthase. Mol Biol Rep 27: 141–148 jannaschii. Science 273: 1058–1073 Dose MM, Hirasawa M, Kleis-SanFrancisco S, Lew EL and Carlberg I and Nordlund S (1991) Purification and partial Knaff DB (1997) The ferredoxin-binding site of ferre- characterization of glutamate synthase from Rhodospirillum doxin:nitrite oxidoreductase. Differential chemical modifica- 213

tion of the free enzyme and its complex with ferredoxin. of GS/GOGAT cycle and GDH in ammonium and amino Plant Physiol 114: 1047–1053 acids metabolism in germinating Medicago truncatula seeds. Doverskog M, Jacobsson U, Chapman BE, Kuchel PW and Planta (in press) Ha¨ ggstro¨ m L (2000) Determination of NADH-dependent Grumbles RM (1989) Inability of glutamate synthase-deficient glutamate synthase (GOGAT) in Spodoptera frugiperda Arabidopsis thaliana. Plant Physiol Biochem 27: 185–191 (Sf9) insect cells by a selective 1H/5N NMR in vitro assay. Ha¨ usler RE, Blackwell RD, Lea PJ and Leegood RC (1994) J Bacteriol 79: 87–97 Control of photosynthesis in barley leaves with reduced Edwards JW, Walker EL and Coruzzi GM (1990) Cell-specific activities of glutamine synthetase or glutamate synthase. expression in transgenic plants reveals nonoverlapping roles I. Plant characteristics and changes in nitrate, ammonium for chloroplast and cytosolic glutamine synthetase. Proc and amino acids. Planta 194: 406–417 Natl Acad Sci USA 87: 3459–3463 Hayakawa T, Yamaya T, Kamachi K and Ojima K (1992) Elminger MV and Mohr H (1991) Coaction of blue/ultravi- Purification, characterization, and immunological properties olet-A light and light absorbed by phytochrome in of NADH-dependent glutamate synthase from rice cell controlling the appearance of ferredoxin-dependent gluta- cultures. Plant Physiol 98: 1317–1322 mate synthase in Scot pine (Pinus sylvestris L.) seedlings. Hayakawa T, Nakamura T, Hattori F, Mae T, Ojima K and Planta 183: 374–380 Yamaya T (1994) Cellular localization of NADH-dependent Ely B, Amarasinghe ABC and Bender RA (1978) Ammonia glutamate synthase protein in vascular bundles of unex- assimilation and glutamate formation in Caulibacter cres- panded leaf blades and young grains of rice plants. Planta centus. J Bacteriol 133: 225–230 193: 455–460 Ferrario-Me´ry S, Suzuki A, Kunz C, Valadier MH, Roux Y, Hemmila¨ IA and Ma¨ ntsa¨ la¨ P (1978) Purification and properties Hirel B and Foyer CH (2000) Modulation of amino acid of glutamate synthase and glutamate dehydrogenase from metabolism in transformed tobacco plants deficient in Bacillus magaterium. Biochem J 173, 45–52 Fd-GOGAT. Plant Soil 221: 67–79 Hirasawa M and Knaff DB (1993) The role of lysine and Ferrario-Me´ry S, Valadier MH, Godfroy N, Miallier D, Hirel arginine residues at the ferredoxin-binding site of spinach B, Foyer CH and Suzuki A (2002) Diurnal changes in glutamate synthase. Biochim Biophys Acta 1144: 85–91 ammonia assimilation in transformed tobacco plants Hirasawa M, Boyer JM, Gray KA, Davis DJ and Knaff DB expressing ferredoxin-dependent glutamate synthase mRNA (1986) The interaction of ferredoxin with chloroplast ferre- in the antisense orientation. Plant Sci 112: 524–530 doxin-linked enzymes. Biochim Biophys Acta 851: 23–28 Filetici P, Martegani MP, Valenzuela L, Gonza´lez A and Hirasawa M, Boyer MJ, Gray KA, Davis DJ and Knaff DB Ballario P (1996) Sequence of the gene from Saccharomyces (1987) The interaction of ferredoxin-linked sulfite reductase cerevisiae reveals the domain structure of yeast glutamate with ferredoxin. FEBS Lett 221: 343–348 synthase. Yeast 12: 1359–1366 Hirasawa M, Droux M, Gray KA, Boyer JM, Davis DJ, Fox GG, Ratcliffe RG, Robinson SA and Stewart GR (1995) Buchanan BB and Knaff DB (1988) Ferredoxin-thioredoxin Evidence for deamination by glutamate-dehydrogenase in reductase: Properties of its complex with ferredoxin. Biochim higher plants. Can J Bot 73: 1112–1115 Biophys Acta 935: 1–8 GalvańF,Ma´rquez J and Vega JM (1984) Purification and Hirasawa M, Chang K-T, Morrow KJ and Knaff DB (1989) molecular properties of ferredoxin-glutamate synthase from Circular dichroism, binding and immunological studies of Chlamydomonas reinhardii. Planta 162: 180–187 the interaction between spinach ferredoxin and glutamate Garcıá-Gutie´rrez A, Cantoń FR, Gallardo F, Sańchez-Jimeńez synthase. Biochim Biophys Acta 977: 150–156 F and Cańovas FM (1995) Expression of ferredoxin-depen- Hirasawa M, Chang K-T and Knaff DB (1991) The interaction dent glutamate synthase in dark-grown pine seedlings. Plant of ferredoxin and glutamate synthase: Cross-linking and Mol Biol 27: 115–128 immunological studies. Arch Biochem Biophys 286: 171–177 Garcıá-Sańchez MI, Dıáz_Quintana A, Gotor C, Jacquot J-P, Hirasawa M, Robertson DE, Ameyibor E, Johnson MK and De la Rosa M and Vega JM (2000) Homology predicted Knaff DB (1992) Oxidation-reduction properties of the structure and functional interaction of ferredoxin from the ferredoxin-linked glutamate synthase from spinach leaf. eukaryotic alga Chlamydomonas reinhardtii with nitrite Biochim Biophys Acta 1100: 105–108 reductase and glutamate synthase. J Biol Inorg Chem 5: Hirasawa M, Hurley JK, Salamon Z, Tollin G and Knaff DB 713–719 (1996) Oxidation-reduction and transient kinetic studies of Geary LE and Meister A (1977) On the mechanism of spinach ferredoxin-dependent glutamate synthase. Arch glutamine-dependent reductive amination of a-ketoglutarate Biochem Biophys 330: 209–215 catalyzed by glutamate synthase. J Biol Chem 252: 3501–3508 Hirasawa M, Hurley JK, Salamon Z, Tollin G, Markley JL, Glauser DA, Bourquin F, Manieri W and Schu¨ rmann P (2004) Cheng H, Xia B and Knaff DB (1998) The role of aromatic Characterization of ferredoxin:thioredoxin reductase modi- and acidic amino acids in the electron transfer reaction fied by site-directed mutagenesis. J Biol Chem 279: 16662– catalyzed by spinach ferredoxin-dependent glutamate syn- 16669 thase. Biochim Biophys Acta 1363: 134–136 Goto S, Akagawa,T, Kojima S, Hayakawa T and Yamaya T Hirasawa M, Rubio LM, Griffin JL, Flores E, Herrero A, Li J, (1998) Organisation and structure of NADH-dependent Kim S-K, Hurley JK, Tollin G and Knaff DB (2004) glutamate synthase from rice plants. Biochim Biophys Acta Complex formation between ferredoxin and Synechococcus 1387: 298–308 ferredoxin:nitrate oxidoreductase. Biochim Biophys Acta Gregerson RG, Miller SS, Twary SN, Gantt JS and Vance CP 1608: 155–162 (1993) Molecular characterization of NADH-dependent glu- Hirayama C, Saito H, Konno K and Shinbo H (1998) tamate synthase from Alfalfa nodules. Plant Cell 5: 215–226 Purification and characterization of NADH-dependent Grevarec G, Bouton S, Jaspard E, Riou M-T, Cliquet J-B, glutamate synthase from the silkworm fat body (Bombyx Suzuki A, Anis M and Limami AM (2004) Respective roles mori). Insect Biochem Mol Biol 28: 473–482 214

Hirose N and Yamaya T (1999) Okadaic acid mimics nitrogen- glutamate synthase from the hyperthermophilic archaeon stimulated transcription of the NADH-glutamate synthase Pyrococcus sp. KOD1. Mol Gen Genet 254: 635–642 gene in rice cell culture. Plant Physiol 121: 805–812 Joy KW, Blackwell RD and Lea PJ (1992) Assimilation of Holden HM, Jacobsen BL, Hurley JK, Tollin G, Oh B-H, nitrogen in mutants lacking enzymes of the glutamate Skjeldal L, Chae YK, Cheng H, Xia B and Markley JL synthase cycle. J Exp Bot 43: 139–145 (1994) Structure-function studies of [2Fe-2S] ferredoxins. Kaneko T, Sato S, Kotani H, Tanaka A, Asamizu E, J Bioenerg Biomembr 26: 67–88 Nakamura Y, Miyajima N, Hirosawa M, Sugiura M, Hsieh MH, Lam H-M, van de Loo FJ and Coruzzi G (1998) A Sakamoto S, Kimura T, Hosouchi T, Matsuno A, Muraki PII-like protein in Arabidopsis: Putative role in nitrogen A, Nakazaki N, Naruo K, Okumura S, Shimpo S, Takeuchi sensing. Proc Natl Acad Sci USA 95: 13965–13970 C, Wada T, Watanabe A, Yamada M, Yasuda M and Hummlet J and Mora J (1980) Regulation and function of Tabata S (1996) Sequence analysis of the genome of the glutamate synthase. Biochem Biophys Res Commun 96: unicellular cyanobacterium Synechocystis sp. strain 1688–1694 PCC6893. II. Sequence determination of the entire genome Hurley JK, Cheng H, Xia B, Markley JL. Medina M, Gomez- and assignment of potential protein coding regions. DNA Moreno C and Tollin G (1993) An aromatic amino acid is Res 3: 109–136 required at position 65 in Anabaena ferredoxin for rapid Kawarabayashi Y, Sawada M, Horikawa H, Haikawa Y, electron transfer to ferredoxin:NADP+ reductase. J Am Hino Y, Yamamoto S, Sekine M, Baba S, Kosugi H, Chem Soc 115: 11698–11701 Hosoyama A, Nagai Y, Sakai M, Ogura K, Otsuka R, Hurley JK, Fillat MF, Go´mez-Moreno C and Tollin G (1996) Nakazawa H, Takamiya M, Ohfuku Y, Funahashi T, Electrostatic and hydrophobic interactions during complex Tanaka T, Kudo Y, Yamazaki J, Kushida N, Oguchi A, formation and electron transfer in the ferredoxin/ferre- Aoki K, Yoshizawa T, Nakaamura Y, Robb FT, Horikoshi doxin:NADP+ reductase system from Anabaena.JAm K, Masuchi Y, Shizuya H and Kikuchi H (1998) Complete Chem Soc 118: 5526–5531 sequence and gene organization of the genome of a hyper- Hurley JK, Weber-Main AM, Stankovich MT, Benning MM, thermophilic archaebacterium, Pyrococcus horikoshii OT3. Thoden JB, Vanhooke JL, Holden HM, Chae YK, Xia B, DNA Res 5: 55–76 Cheng H, Markley JL, Martinez-Ju´lvez M, Go´mez-Moreno Kendall AC, Wallsgrove RM, Hall NP, Turner JC and Lea PJ C, Schmeits JL and Tollin G (1997) Structure–function (1986) Carbon and nitrogen metabolisms in barley (Hordeum relationships in Anabaena ferredoxin: correlations between vulgare L.) mutants lacking ferredoxin-dependent glutamate X-ray structures, reduction potentials, and rate constants of synthase. Planta 168: 316–323 electron transfer to ferredoxin:NADP+ reductase for site- Klenk H-P, Clayton R, Tomb J-F, Whaite O, Nelson KE, specific ferredoxin mutants. Biochemistry 36: 11100–11117 Ketchum KA, Dodson RJ, Gwinn M, Hickey EK, Peterson Ireland RJ and Lea PJ (1999) The enzymes of glutamine, J, Richardson DL, Kerlavage AR, Graham DE, Krypides glutamate, asparagine, and aspartate metabolism. In: NC, Fleischmann RD, Quaxkenbush J, Lee NH, Sutton Singh BK (ed) Plant Amino Acids. Biochemistry and GG, Gill S, Kirkness EF, Dougherty BA, McKenny K, Biotechnology, pp 49–109. American Cyanamid Company, Adams MD, Loftus B, Peterson S, Reich CI, McNeil LK, Princeton New Jersey/Marcel Dekker, New York Badger JH, Glodek A, Zhou L, Overbeek R, Gocayane JD, Ishiyama K, Hayakawa T and Yamaya T (1998) Expression of Weidman JF, McDonald L, Utterback T, Cotton MD, NADH-dependent glutamate synthase protein in the epider- Spriggs T, Artiach P, Kaine BP, Sykes SM, Sadow PW, mis and epidermis of rice roots in response to the supply of D’Andrea KP, Bowman C, Fujii C, Garland SA, Mason nitrogen. Planta 204: 288–294 TM, Olsen GJ, Fraser CM, Smith HO, Woese CR and Ishiyama K, Kojima S, Takahashi H, Hayakawa T and Venter JC (1997) The compete genome sequence of the Yamaya T (2003) Cell type distinct accumulation of mRNA hyperthermophilic, sulphate-reducing archaeon Archaeoglo- and protein for NADH-dependent glutamate synthase in rice bus fulgidus. Nature 390: 364–370 + roots in response to the supply of NH4 . Plant Physiol Knaff DB (1996) Ferredoxin and ferredoxin-dependent en- Biochem 41: 643–647 zymes. In: Ort DR, Yocum CF (eds) Oxygenic Photosyn- Jelesarov I and Bosshard HR (1994) Thermodynamics of thesis. The Light Reactions, pp 333–361. Kluwer Academic ferredoxin binding to ferredoxin:NADP+ reductase and the Publishers, Dordrecht, The Netherlands role of water at the complex interface. Biochemistry 33: Knaff DB, Hirasawa M, Ameyibor E, Fu W and Johnson MK 13321–13328 (1991) Spectroscopic evidence for a [3Fe-4S] cluster in Jelesarov I, De Pascalis AR, Koppenol WH, Hirasawa M, spinach glutamate synthase. J Biol Chem 266: 15080–15084 Knaff DB and Bosshard HR (1993) Ferredoxin binding site Kojima S, Kimura, M, Nozaki Y and Yamaya T (2000) on ferredoxin:NADP+ reductase. Differential chemical Analysis of a promoter for the NADH-glutamate synthase modification of free and ferredoxin-bound enzyme. Eur J gene in rice (Oryza sativa): cell-type specific expression in Biochem 216: 57–66 developing organs of transgenic rice plants. Aust J Plant Jeschke WD and Pate JS (1991) Modeling of the partitioning, Physiol 27: 787–793 assimilation and storage of nitrate within root and shoot Kurisu G, Kusunoki M, Katoh E, Yamazaki T, Teshima K, organs of castor bean (Ricinus communis L.). J Exp Bot 42: Onda Y, Kimata-Ariga Y and Hase T (2001) Structure of 1091–1103 the electron transfer complex between ferredoxin and Jiang P and Ninfa AJ (1999) Regulation of autophosphoryla- ferredoxin-NADP+ reductase. Nat Struct Biol 2: 117–121 tion of Escherichia coli nitrogen regulator II by the PII signal Lam H-M, Peng S-SY and Coruzzi GM (1994) Metabolic transduction protein. J Bacteriol 181: 1906-1911 regulation of the gene encoding glutamine-dependent Jongsareejit B, Rahman RNZA, Fujiwara S and Imanaka T asparagine synthetase in Arabidopsis thaliana. Plant Physiol (1997) Gene cloning, sequencing and enzymatic properties of 106: 1364–1357 215

Lam H-M, Coschigano KT, Oliveira IC, Melo-Oliveira R and Proceeding: Plant Biology and the Challenge of Functional Coruzzi GM (1996) The molecular-genetics of nitrogen Genomics, p 60. 5e` me Colloque Geńe´ral de la Socie´te´ assimilation into amino acids in higher plants. Annu Rev Française de Physio1ogie Ve´ge´tale. Universite´de Paris 11, Plant Physiol Plant Mol Biol 47: 569–593 Orsay, France Lancien M, Martin M, Hsieh M-H, Leustek T, Goodman H Miflin BJ and Habash DZ (2002) The role of glutamine and Coruzzi GM (2002) Arabidopsis glt-T mutant defines a synthetase and glutamate dehydrogenase in nitrogen assim- role for NADH-GOGAT in the non-photorespiratory ilation and possibilities for improvement in the nitrogen ammonium assimilatory pathway. Plant J 29: 347–358 utilization of crops. J Exp Bot 53: 979–987 Lea PJ and Miflin BJ (2004) Glutamate synthase and Migge A and Becker TW (1996) In tobacco leaves, the genes synthesis of glutamate in plants. Plant Physiol Biochem encoding the nitrate-reducing or the ammonium-assimilating 41: 555–564 enzymes are regulated differently by external nitrogen Lea PJ, Robinson SA and Stewart GR (1990) The enzymology sources. Plant Physiol Biochem 34: 665–671 and metabolism of glutamine, glutamate, and asparagine. In: Mikami B and Ida S (1989) Spinach ferredoxin-nitrite reduc- Miflin BJ and Lea PJ (eds) The Biochemistry of Plants, Vol tase: Characterization of catalytic activity and interaction of 16, pp 121–159. Academic Press, New York the enzyme with substrates. J Biochem 105: 47–50 Loulakakis KA and Roubelakis-Angelakis KA (1997) Molec- Millar AJ, Straume M, Chory J, Chua N-H and Kay SA (1995) ular cloning and characterization of cDNAs coding for The regulation of circadian period by phototransduction ferredoxin-dependent glutamate synthase from Vitis vinifera. pathways in Arabidopsis. Science 267: 1163–1166 Physiol Plant 101: 220–228 Morales R, Charon M-H, Kachalova G, Serre L, Medina M, Madonna MJ, Fuchs RL and Brenchley JE (1985) Fine Go´mez-Moreno C and Frey M (2000) A redox-dependent structure analysis of Salmonella typhimurium glutamate interaction between two electron-transfer partners involved synthase gene. J Bacteriol 161: 353–360 in photosynthesis. EMBO Rep 1: 271–276 Magalha˜ es JR, Ju GC, Rich PJ and Rhodes D (1990) Kinetics Murray DM and Kennedy IR (1980) Changes in activities of 15 + of NH4 assimilation in Zea mays. Preliminary studies enzymes of nitrogen metabolism in seedcoats and cotyledons with a glutamate dehydrogenase (GDH1) null mutant. Plant during embryo development in pea seeds. Plant Physiol 66: Physiol 94: 647–656 782–786 Marque´s S, Florencio FJ and Candau P (1992) Purification and Nagatani H, Shimizu M and Valentine RC (1971) The characterization of the ferredoxin-glutamate synthase from mechanism of ammonia assimilation in nitrogen fixing the unicellular cyanobacterium Synechococcus sp. PCC 6301. bacteria. Arch Mikrobiol 79: 164–175 Eur J Biochem 206: 69–77 Nalbantoglu B, Hirasawa M, Moomaw C, Nguyen H, Knaff Ma´rquez AJ, Galvań F and Vega JM (1984) Purification and DB and Allen R (1994) Cloning and sequencing of the gene characterization of the NADH-glutamate synthase from encoding spinach ferredoxin-dependent glutamate synthase. Chlamydomonas reinhardii. Plant Sci Lett 34: 305–314 Biochim Biophys Acta 1183: 557–561 Martıń-Figueroa E, Navarro F and Florencio FJ (2000) The Navarro F, Cha´vez S, Candau P and Florencio FJ (1995) GS-GOGAT pathway is not operative in the heterocists. Existence of two ferredoxin-glutamate synthases in the Cloning and expression of glsF gene from the cyanobacte- cyanobacterium Synechocystis sp. PCC 6803. Isolation and rium Anabaena sp. PCC 7120. FEBS Lett 476: 282–286 insertional inactivation of gltB and gltS genes. Plant Mol Matt P, Geiger M, Walch-Liu, Engels C, Krapp A and Stitt M Biol 27: 753–767 (2001) The immediate cause of the diurnal changes of Navarro F, Martin-Figueroa E, Candau P and Florencio FJ nitrogen metabolism in leaves of nitrate-replete tobacco: a (2000) Ferredoxin-dependent iron-sulfur flavoprotein from major imbalance between the rate of nitrate reduction and cyanobacterium Synechocystis sp. PCC 6803: Expression and the rates of nitrate uptake and ammonium metabolism assembly in Escherichia coli. Arch Biochem Biophys 379: during the first part of the light period. Plant Cell Environ 267–276 24: 177–190 Neff MM, Fankhauser C and Chory J (2000) Light: an Mattana M, Coraggio I, Brambilla I, Bertani A and Reggiani R indicator of time and place. Genes Dev 14: 553–590 (1996) Nitrate assimilation during the anaerobic germination Oaks A, Jones K and Misra S (1979) A comparison of of rice: Expression of ferredoxin-dependent glutamate syn- glutamate synthase obtained from maize endosperms and thase. Planta 199: 74–78 roots. Plant Physiol 63: 793–795 McKenzie GH, Ch’Ng AL and Gayler KR (1979) Glutamine Okon Y, Albrecht SL and Burris RH (1976) Carbon and synthetase/glutamine:a-ketoglutarate aminotransferase in ammonia metabolism of Spirillum lipoferum. J Bacteriol 128: chloroplasts from the marine alga Caulerpa simpliciuscula. 592–597 Plant Physiol 63: 578–582 Okuhara H, Matsumura T, Fujita Y and Hase T (1999) Cloning McNeil DL, Atkins CA and Pate JS (1979) Uptake and and inactivation of genes encoding ferredoxin-and NADH- utilization of xylem borne amino compounds by shoot dependent glutamate synthases in the cyanobacterium Plec- organs of a legume. Plant Physiol 63: 1076–1081 tonema boryanum. Imbalances of nitrogen and carbon Melo-Olveira R, Oliveira IC and Coruzzi G (1996) Arabidopsis assimilations caused by deficiency of the ferredoxin-depen- mutant analysis and gene regulation define a non-redundant dent enzyme. Plant Physiol 120: 33–42 role for glutamate dehydrogenase in nitrogen assimilation. Oliveira IC and Coruzzi GM (1999) Carbon and amino acids Proc Natl Acad Sci USA 93: 4718–4723 reciprocally modulate the expression of glutamine synthetase Meyer C, Roussel H, Thibault C, Gaudon V, Dorbe M-F, in Arabidopsis. Plant Physiol 121: 301–309 Alboresi A, Truong H-N, Brun F, Zobolowska L, Krapp A Oliveira IC, Lam H-M, Coschigano K, Melo-Oliveira R and and Vedele FR (2003) Regulation of the Arabidopsis Coruzzi GM (1997) Molecular-genetic dissection of transcriptome by variations in nitrogen supply. In Prioul ammonium assimilation in Arabidopsis thaliana. Plant Phys- J-L, Vidal J, Brulfert J, Miginiac-Maslow M (eds) iol Biochem 35: 185–198 216

Oliver G, Gosset G, Sanchez-Pescador R, Lzoya E, Ku LM, Sakakibara H, Watanabe M, Hase T and Sugiyama T (1991) Florez N, Becerril B and Valle F (1987) Determination of the Molecular cloning and characterization of complementary nucleotide sequence for the glutamate synthase structural DNA encoding for ferredoxin-dependent glutamate synthase genes of Escherichia coli K-12. Gene 60: 1–11 in maize leaf. J Biol Chem 266: 2028–2035 Pajuelo P, Pajuelo E, Forde B and Ma´rquez AJ (1997) Sakakibara H, Kawabata S, Hase T and Sugiyama T (1992a) Regulation of the expression of ferredoxin-glutamate syn- Molecular cloning of the family of glutamine synthetase thase in barley. Planta 203: 517–525 genes from maize. Expression of genes for glutamine Pate JS (1980) Transport and partitioning of nitrogen solutes. synthetase and ferredoxin-dependent glutamate synthase in Annu Rev Plant Physiol 31: 313–340 photosynthetic and non-photosynthetic tissues. Plant Cell Pate JS and Layzell DB (1990) Energetics and biological costs Physiol 33: 49–58 of nitrogen assimilation. In: Miflin BJ and Lea PJ (eds) The Sakakibara H, Kawabata S, Hase T and Sugiyama T (1992b) Biochemistry of Plants, pp 1–42. New York, Academic Press Differential effects of nitrate and light on the expression of Pelanda R, Vanoni MA, Perego M, Piubelli L, Galizzi A, Curti glutamine synthetases and ferredoxin-dependent glutamate B and Zanetti G (1993) Glutamate synthase genes of the synthase in maize. Plant Cell Physio1 33: 1193–1198 diazotroph Azospirillum brasilense. Cloning, sequencing, and Sakakibara H, Kobayashi K, Deji A and Sugiyama T (1997) analysis of functional domains. J Biol Chem 268: 3099–3106 Partial characterization of the signaling pathway for the Peterman TK and Goodman HM (1991) The glutamine nitrate-dependent expression of genes for nitrogen-assimila- synthetase gene family of Arabidopsis thaliana: light-regulatory enzymes using detached maize leaves. Plant Cell Physiol tion and differential expression in leaves, roots and seeds. 38: 837–843 Mol Gen Genet 230: 145–154 Scheible W-R, Gonza´lez-Fontes A, Lauerer M, Mu¨ ller-Ro¨ ber Petoukhov MV, Svergun DI, Kornarev PV, Ravasio S, van B, Caboche M and Stitt M (1997) Nitrate acts as a signal to den Heuvel RHH, Curtis B and Vanoni M (2003) Quater- induce organic acid metabolism and repress starch metab- nary structure of Azospirillum brasilense NADPH-depen- olism in tobacco. Plant Cell 9: 783–798 dent glutamate synthase in solution as revealed by Scheible W-R, Krapp A and Stitt M (2000) Reciprocal diurnal synchrotron radiation X-ray scattering. J Biol Chem 278: changes of phosphoenolpyruvate carboxylase expression and 29933–29939 cytosolic pyruvate kinase, citrate synthase and NADP- Petracek M, Dickey LF, Nguyen TT, Gatz C, Sowinsky DA, isocitrate dehydrogenase expression regulate organic acid Allen GC and Thompson WF (1998) Ferredoxin-l mRNA is metabolisms during nitrate assimilation in tobacco leaves. stabilized by changes in photosynthetic electron transport. Plant Cell Environ 23: 1155–1167 Proc Natl Acad Sci USA 95: 9009–9013 Schmitz S, Navarro F, Kutzki CK, Florencio FJ and Bo¨ hme H Ravasio S, Curti B and Vanoni MA (2001) Determination of (1996) Glutamate 94 of [2Fe-2S]-ferredoxins is important for the midpoint potential of the FAD and FMN flavin efficient electron transfer in the 1: 1 complex with ferredoxin- cofactors and of the 3Fe/4S cluster of glutamate synthase. glutamate synthase (GltS) from Synechocystis sp. PCC 6803. Biochemistry 40: 5533–5541 Biochem Biophys Acta 1277: 135–140 Ravasio S, Dossena L, Martin-Figueroa E, Florencio FJ, Schoenbeck MA, Temple SJ, Trepp GB, Blumenthal JM, Samac Mattevi A, Morandi P, Curti B and Vanoni MA (2002) DA, Gantt JS, Hermandez G and Vance CP (2000) Decreased Properties of recombinant ferredoxin-dependent glutamate NADH-glutamate synthase activity in nodules and flowers of synthase of Synechocystis PCC6803. Comparison with the alfalfa (Medicago sativa L.) transformed with an antisense Azospirillum brasilense NADPH-dependent enzyme and its glutamate synthase transgene. J Exp Bot 51: 29–39 isolated a subunit. Biochemistry 41: 8120–8133 Seshachalam RV, Subramanyama MVV and Krishnamoorthy Ratcliffe RG and Shachar-Hill Y (2001) Probing plant metab- RV (1992) New pathway of utilization of ammonia nitrogen olism with NMR. Annu Rev Plant Physiol Plant Mol Biol for the synthesis of amino acids through NADH dependent 52: 499–526 transaminases in Mombyx mori L. Physiol Entomol 17: Redinbaugh MG and Campbell WH (1993) Glutamine synthe- 281–287 tase and ferredoxin-dependent glutamate synthase expres- Smith CJ, Hespell RB and Bryan MP (1977) Ammonia sion in the maize (Zea mays) root primary response to assimilation and glutamate formation in the anaerobe nitrate. Plant Physiol 101: 1249–1255 Selenomonas ruminantium. J Bacteriol 141: 593–602 Reith M and Munholland J (1993) A high-resolution gene map Smith DR, Doucette-Stamm LA, Deloughery C, Lee H, Dubois of the chloroplast genome of the red alga Porphyra purpurea. J, Aldredge T, Bashirzadeh R, Blakely D, Cook R, Gilbert Plant Cell 5: 465–475 K, Harrison D, Hoang L, Keagle P, Lumm W, Pothier B, Reitzer LJ (1996) Ammonia assimilation and the biosynthesis Qiu D, Spadafora R, Vicaire R, Wang Y, Wierzbowski J, of glutamine, glutamate, aspartate, asparagine, L-alanine Gibson R, Jiwani N, Caruso A, Bush D, Safer H, Patwell D, and D-alanine. In: Neidhart FC (ed) Escherichia coli and Prabhakar S, McDougall S, Shimer G, Goyal A, Pietrokov- Salmonella: Cellular and Molecular Biology, pp 391–407. ski S, Church GM, Daniels CJ, Mao J-I, Rice P, No¨ lling J ASM Press, Washington, DC and Reeve JN (1997) Complete genome sequence of Romero M, Guzman-Leoń S, Aranda C, Gonza´lez-Halphen D, Methanobacterium thermoautotrophiocum DH: functional Valenzuela L and Gonza´lez A (2000) Pathway for glutamate analysis and comparative genomics. J Bacteriol 179: 7135– synthesis in the yeast Kluyveromyces lactis. Microbiol 146: 7155 239–245 Somers DE, Devlin PF and Kay SA (1998) Phytochromes and Rypniewski WR, Breiter DR, Benning MM, Wesenberg G, Oh cryptochromes in the entrainment of the Arabidopsis circa- B-H, Markley JL, Rayment I and Holden HM (1991) dian clock. Science 282: 1488–1490 Crystallization and structure determination to 2.5-A˚ resolu- Somerville CR and Ogren WL (1980) Inhibition of photosyn- tion of the oxidized [2Fe-2S] ferredoxin isolated from thesis in Arabidopsis mutants lacking leaf glutamate syn- Anabaena 7120. Biochemistry 30: 4126–4131 thase activity. Nature 286: 257–259 217

Staigner D and Apel K (1998) Circadian clock-regulated van den Heuvel RHH, Svergun DI, Petoukhov MV, Coda A, expression of an RNA-binding protein in Arabidopsis: Curti B, Ravasio S, Vanoni MA and Mattevi A (2003) The characterization of a minimal promoter element. Mol Gen active conformation of glutamate synthase and its binding to Genet 261: 811–819 ferredoxin. J Mol Biol 330: 113–128 Stitt M, Mu¨ ller C, Matt P, Gibon Y, Carillo P, Morcuende R, van den Heuvel RHH, Curti B, Vanoni MA and Mattevi A Scheible W-R and Krapp A (2002) Steps towards an (2004) Glutamate synthase: a fascinating pathway from l- integrated view of nitrogen metabolism. J Exp Bot 53: glutamine to l-glutamate. Cell Mol Life Sci 61: 669–681 959–970 Vance CP, Gregerson RG, Robinson DL, Miller SS and Gantt Suzuki A and Rothstein S (1997) Structure and regulation of JS (1994) Primary assimilation of nitrogen in alfalfa nodules: ferredoxin-dependent glutamate synthase from Arabidopsis molecular features of the enzymes involved. Plant Sci 101: thaliana. Cloning of cDNA, expression in different tissues of 51–64 wild-type and gltS mutant strains, and light induction. Eur J Vance CP, Miller SS, Gregerson RG, Samac DA, Robinson DL Biochem 243: 708–718 and Gantt S (1995) Alfalfa NADH-dependent glutamate Suzuki A, Nato A and Gadal P (1982) Glutamate synthase synthase: structure of the gene and importance in symbiotic isoforms in tobacco cultured cells. Plant Sci Lett 33: 93– N2 fixation. Plant J 8: 345–358 101 Vanoni MA and Curti B (1999) Glutamate synthase: a complex Suzuki A, Oaks A, Jacquot J-P, Vidal J and Gadal P (1985) An iron-sulfur flavoprotein. Cell Mol Life Sci 55: 617–638 electron donor transport system in maize roots for reactions Vanoni MA, Negri A, Zanetti G, Ronchi S and Curti B (1990) of glutamate synthase and nitrite reductase. Physiological Structural studies on the subunits of glutamate synthase and immunochemical properties of the electron carrier and from Azospirillum brasilense. Biochim Biophys Acta 1039: pyridine nucleotide reductase. Plant Physiol 78: 374–378 374–377 Suzuki A, Burkhart W and Rothstein S (1996) Nitrogen effects Vanoni MA, Fischer F, Ravasio S, Verzotti E, Edmondson DE, on the induction of ferredoxin-dependent glutamate synthase Hagen WR, Zanetti G and Curti B (1998) The recombinant and its mRNA in maize leaves under the light. Plant Sci 114: a subunit of glutamate synthase: spectroscopic and catalytic 83–91 properties. Biochemistry 37: 1828–1838 Suzuki A, Rioual S, Godfroy N, Roux Y, Boutin JP and Vanoni M, Dossena L, van den Heuvel RHH and Curti B Rothstein S (2001) Regulation by light and metabolites of (2005) Structure–function studies on the complex iron–sulfur ferredoxin-dependent glutamate synthase in maize. Physiol flavoprotein glutamate synthase: the key enzyme of ammo- Plant 112: 524–530 nia assimilation. Photosynthe Res 83: 219–238 (this issue) Taira M, Valtersson U, Burkhardt B and Ludwig RA (2004) Vigara AJ, Garcıá-Sańchez MI, Gotor C and Vega JM Arabidopsis thaliana GLN2-encoded glutamine synthetase is (1996) Interaction between glutamate synthase and ferre- dual targeted to leaf mitochondria and chloroplasts. Plant doxin from Monoraphidium braunii. Chemical modifica- Cell 16: 2048–2058 tions and cross-linking studies. Plant Physiol Biochem 34: Teller S, Schmidt KH and Appenroth KJ (1996) Ferredoxin- 707–711 dependent but not NADH-dependent glutamate synthase is Watanabe S, Sakai T, Goto S, Yaginuma T, Hayakawa T and regulated by phytochrome and a blue/UV-A light receptor in Yamaya T (1996) Expression of NADH-dependent gluta- turions of Spirodela polyrhiza. Plant Physiol Biochem 34: mate synthase in response to the supply of nitrogen in rice 713–719 cells in suspension culture. Plant Cell Physiol 37: 1034–1037 Thum KE, Shasha DE, Lajay LV and Coruzzi GM (2003) Yamaya T, Hayakawa T, Tanasawa K, Kamachi K, Mae T and Light- and carbon-signaling pathways. Modeling circuits of Ojima K (1992) Tissue distribution of glutamate synthase interactions. Plant Physiol 132: 440-452 and glutamine synthetase in rice leaves. Occurrence of Tobin AK and Yamaya T (2001) Cellular compartmentation of NADH-dependent glutamate synthase protein and activity ammonium assimilation in rice and barley. J Exp Bot 52: in the unexpanded non-green leaf blades. Plant Physiol 100: 591–604 1427–1432 Trepp GB, van de Mortel M, Yoshioka H, Miller SS, Samac Yamaya T, Tanno H, Hirose N, Watanabe S and Hayakawa T DA, Gantt JS and Vance CP (1999a) NADH-glutamate (1995) A supply of nitrogen causes increase in the level of synthase in alfalfa root nodules. Genetic regulation and NADH-dependent glutamate synthase protein and in the cellular expression. Plant Physiol 119: 817–828 activity of the enzyme in roots of rice seedlings. Plant Cell Trepp GB, Plank DW, Gantt JS and Vance CP (l999b) NADH- Physiol 36: 1197–1204 glutamate synthase in alfalfa root nodules. Immunological Yonekura-Sakakibara K, Onda Y, Ashikari T, Tanaka Y, localization. Plant Physiol 119: 829–837 Kusumi T and Hase T (2000) Analysis of Reductant Turano FJ and Muhitch MJ (1999) Differential accumulation supply systems for ferredoxin-dependent sulfite reductase of ferredoxin- and NADH- dependent glutamate synthase in photosynthetic and non-photosynthetic organs of maize. activities, peptides, and transcripts in developing soybean Plant Physiol 122: 887–894 seedlings in response to light, nitrogen, and nodulation. Zehnacker C, Becker TW, Suzuki A, Carrayol E, Caboche M Physiol Plant 107: 407–418 and Hirel B (1992) Purification and properties of tobacco Valentin K, Kostrzewa M and Zetsche K (1993) Glutamate ferredoxin-dependent glutamate synthase, and isolation of synthase is plastid-encoded in a red alga: implications for the corresponding cDNA clones. Light-inducibility and organ- evolution of glutamate synthases. Plant Mol Biol 23: 77–85 specificity of gene transcription and protein expression. van den Heuvel RHH, Ferrari D, Bossi RT, Ravasio S, Curti Planta 187: 266–274 B, Vanoni MA, Florencio FJ and Mattevi A (2002) Ziegler C, Feraud M, Jouglet T, Viret L, Spampinato A, Structural studies on the synchronization of catalytic Paganelli V, Ben Hammouda M and Suzuki A (2003) centers in glutamate synthase. J Biol Chem 277: 24579– Regulation of promoter activity of ferredoxin-dependent 24583 glutamate synthase. Plant Physiol Biochem 41: 649–655